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B: Biophysical and Biochemical Systems and Processes

QM/MM Molecular Dynamics Simulations Revealed Catalytic Mechanism of Urease
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The Journal of Physical Chemistry B

Cite this: J. Phys. Chem. B 2022, 126, 10, 2087–2097
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https://doi.org/10.1021/acs.jpcb.1c10200
Published March 3, 2022

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Abstract

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Urease catalyzes the hydrolysis of urea to form ammonia and carbamate, inducing an overall pH increase that affects both human health and agriculture. Inhibition, mutagenesis, and kinetic studies have provided insights into its enzymatic role, but there have been debates on the substrate binding mode as well as the reaction mechanism. In the present study, we report quatum mechanics-only (QM-only) and quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) calculations on urease that mainly investigate the binding mode of urea and the mechanism of the urease-catalyzed hydrolysis reaction. Comparison between the experimental data and our QM(GFN2-xTB)/MM metadynamics results demonstrates that urea hydrolysis via a complex with bidentate-bound urea is much more favorable than via that with monodentate-bound urea for both nucleophilic attack and the subsequent proton transfer steps. We also indicate that the bidentate coordination of urea fits the active site with a closed conformation of the mobile flap and can facilitate the stabilization of transition states and intermediates by forming multiple hydrogen bonds with certain active site residues.

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1. Introduction

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Urease, a nickel-containing metalloenzyme, is found in various organisms such as plants, algae, fungi, and prokaryotes. (1−4) The binuclear Ni center in its active site catalyzes urea hydrolysis to form ammonia (NH3) and carbamate, which spontaneously decomposes into another NH3 molecule and bicarbonate (Scheme 1). (5−9) This enzyme exhibits a rate enhancement of ca. 1014-fold over the uncatalyzed reaction.

Scheme 1

Scheme 1. Urea Hydrolysis Reaction
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The enzymatic hydrolysis of urea leads to an overall pH increase that affects both human health and agriculture. (10−13) The human pathogenic bacterium Helicobacter pylori is classified as the most important risk factor for gastric cancer. The urease produced by H. pylori plays an essential role for the colonization of the stomach by neutralizing the acidic environment. Concerning urease reactions in soils, the NH3 volatilization causes the loss of efficacy of urea fertilizer applications.
To design effective inhibitors of urease, numerous crystal structures have been determined both in the absence and in the presence of inhibitors. (14−24) It has been revealed that two divalent nickel ions in the active site, namely, Ni1 and Ni2, are separated by ca. 3.5 Å and coordinated to two bridging ligands, a hydroxide and a carbamylated lysine denoted as K220*, as illustrated in Figure 1. Two histidine residues (H249 and H275) and a water molecule (W1) are coordinated to Ni1, while two histidines (H137 and H139), a monodentate aspartate acid residue (D363), and a water molecule (W2) are bound to Ni2. These water molecules interact with a nearby water molecule (W3) via a strong hydrogen bond. A consensus regarding the enzymatic reaction has been reached that W1 and W3 are initially displaced and the Lewis acid character of the coordinatively unsaturated Ni1 site enhances the electrophilicity of the carbonyl O atom of urea, leading to the formation of the Ni1–O(urea) bond. However, information from the inhibitor bound crystal structures is not sufficient to assess whether urea binds to the active center in such a monodentate manner or a bidentate complex is formed by further replacement of W2 (Figure 1). Thus, several reaction pathways have been proposed for the subsequent catalytic reaction steps. (9,15,25−27)

Figure 1

Figure 1. Active site of Sporosarcina pasteurii urease (SPU) and two possible substrate binding modes, monodentate 1 and bidentate 2.

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Scheme 2 summarizes possible reaction mechanisms (i)–(iv) proposed so far. Inspired by the crystal structures of native Klebsiella aerogenes (KAU), mutagenesis, and kinetic studies, Hausinger and co-workers suggested that urea binds to the active site in a monodentate manner, referred to herein as mechanism (i). (14,25) In mechanism (i), Ni2 enhances the nucleophilicity of W2 by deprotonation, and the doubly protonated H320 (KAU numbering) acts as a general acid. The reaction can proceed through nucleophilic attack of the formed terminal hydroxide ion on the carbonyl C atom of urea to give a tetrahedral intermediate, followed by a proton transfer from H320 to the urea NH2 group. Ciurli and co-workers alternatively proposed mechanism (ii) starting from a bidentate complex on the basis of the crystal structures of native and inhibitor-bound Sporosarcina pasteurii urease (SPU). (15,19,20,23) The recently determined urea bound SPU complex inhibited by fluoride supports mechanism (ii), (24) in which the bridging hydroxide ion acts as both a nucleophile and a general acid.

Scheme 2

Scheme 2. Previously Proposed Urease Reaction Mechanisms (i)–(iv) and Our Proposed One (v), the Details of Which Are Described in the Text, with Amino Acid Numbering for SPU/KAU
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It should be noted that both the steric structures and the active site environments of SPU and KAU are similar, though the protonation state of H323/H320 (SPU/KAU numbering) and positions of nearby residues are different due to the different pH conditions for crystallization. A mixture of mechanisms (i) and (ii) has also proposed as mechanism (iii), in which the bridging hydroxide ion is a nucleophile and H323/H320 acts as a general acid. (26)
In analogy with other metalloenzymes containing only nickel, (28−31) a few computational investigations have also been reported with active site cluster models. (32−34) Suárez et al. (32) have performed density functional theory (DFT) calculations on small active site models to determine the most likely reaction mechanism. They have compared a variant of mechanism (i), where the terminal OH is replaced by H2O with mechanism (ii), and concluded that mechanism (ii) is favorable on the basis of computed barrier heights. Apart from mechanisms involving nucleophilic attack, Estiu and Merz have proposed an elimination mechanism (iv) (33) as with a biomimetic study, (35) but a cyanate intermediate has never been experimentally identified in the catalytic process. In this way, these previous computational studies did not attempt to compare four mechanisms (i)–(iv) explicitly. One must also keep in mind that it is essential that the effect of protein environment on the catalytic activity is taken into account explicitly, not to mention active site residues such as H323/H320.
Here, quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations (36,37) were applied to avoid a pitfall of interpretation arising from the use of small cluster models. The present study focuses on the binding mode of urea to the dinickel center and reinvestigation of the urease-catalyzed reaction by comparing four mechanisms (i)–(iv). We wish to settle a long-standing controversy, providing mechanistic insights into the urease-catalyzed reaction.

2. Computational Method

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2.1. System Setup

To prepare monodentate (1) and bidentate (2) complexes shown in Figure 1, the X-ray crystal structure of the SPU-urea complex refined at a resolution of 1.42 Å was taken from the Protein Data Bank (PDB code: 6QDY). (24) We replaced the bridging fluoride in 6QDY with hydroxide ion and constructed an active site model for 2 consisting of urea, Ni1, Ni2, a bridging hydroxide ion, and side chains of residues coordinated to two Ni2+ ions (Figure S1). A monodentate counterpart with a terminal hydroxide ion (1) was also constructed to obtain appropriate initial positions of urea and the terminal hydroxide ion. All QM calculations were performed with the ORCA 4.2.1 program package. (38) The geometries of the two active site models were fully optimized in the open-shell singlet state using the Gaussian version of UB3LYP method by using the UKS B3LYP/G keyword, together with the RIJCOSX approximation, the def2-SVP basis set, and the auxiliary def2/J basis set. (39−44) Then, their atomic charges were obtained with the ChelpG procedure (45) on the optimized structures (Figure S1). The CHARMM force field parameters for urea and K220* were generated by the CHARMM General Force Field program. (46) The Lennard-Jones parameters proposed by Merz et al. was assigned to Ni2+ ions. (47) The created topology and parameter files were presented in the Supporting Information. Hydrogen atoms were added by the CHARMM-GUI input generator, (48) provided that the epsilon position of H222 was protonated for both 1 and 2, and H323 was doubly protonated for 1. The two systems were in turn solvated within a 100 × 110 × 100 Å rectangular box of water molecules and neutralized by Na+ ions using the Visual Molecular Dynamics (VMD) program, (49) respectively.

2.2. Classical MD and QM/MM MD Equilibration

For 1 and 2, each QM region contained the aforementioned active site complex, combined with A366 and side chains of H222 and H323, while the MM region was defined as the rest of the system as illustrated in Figure 1. Note that A366 and H222 were assumed to form hydrogen-bonding interactions with urea, stabilizing substrate binding and transition states. (9,50) Classical 10 ns MD simulations were performed in the NPT ensemble at 300 K with a time step of 2.0 fs, using the NAMD program. (51) Nonbonded interactions were cutoff at 14 Å with a switching function, and electrostatic interactions were evaluated by the particle mesh Ewald method. (52) During the MD simulations, all the atoms in the QM region represented by the CHARMM General Force Field were virtually kept fixed with the constraintScaling of 50.0. The CHARMM36 force field and TIP3P water models were utilized to describe the MM region. (53) The computed trajectories were listed in Figure S2. The QM/MM MD simulations were performed with NAMD. The tight-binding GFN2-xTB method (54) was employed for the QM region using the ORCA 4.2.1 program package, while the MM subsystem was treated by the same force field parameters as the classical MD simulations. The NAMD-ORCA interface (55) was exploited to compute the forces on the MM atoms. The QM-MM covalent boundary was treated by the link atom method. The total charge and spin multiplicity were set to 1 and 5 for 1 and 0 and 5 for 2 (see below). The two systems were equilibrated without any constraints for 2.0 ps.

2.3. QM-Only Cluster Calculations

Although broken-symmetry DFT calculations with localized basis sets may be useful for investigating urea hydrolysis catalyzed by the dinickel complex with an open-shell singlet ground state, the free energy analysis is still costly for the QM region sizes considered in this work (Figure 1). Alternatively, we chose to use GFN2-xTB, which is fast and can reasonably be accurate for transition metal complexes. (56)
We began by assessing whether the method can be alternative to broken-symmetry DFT calculations. The accuracy of GFN2-xTB for geometry optimization and the energetics of urea hydrolysis was validated by means of smaller cluster models for 1 and 2. For comparison purposes, we used the UB3LYP/def2-SVP method as the reference method on the basis of previous studies of the structural and magnetic properties of the urease active site. (32,34,57,58) An active site model without urea (3) was also tested in the case of a coordinatively unsaturated metal center. To minimize artificial intramolecular interactions, we constructed three QM-only cluster models 1′-3′ in which the side chains of K220* and histidine residues were truncated as methylcarbamic acid and imidazole, respectively (Figure S3). The geometries for 1′–3′ were fully optimized in the open-shell singlet state at the UB3LYP level. In the case of GFN2-xTB, both the quintet and closed-shell singlet states were tested for the optimizations because it gave only closed-shell configurations for the singlet. Then, the potential energy profiles of possible mechanisms starting with 1′ and 2′ were predicted by GFN2-xTB and UB3LYP/def2-SVP. All GFN2-xTB calculations were performed with the ORCA 4.2.1. (38) The Gaussian16 program package (59) was applied to the UB3LYP/def2-SVP method for the purpose of locating saddle points efficiently. Note that we verified that the UB3LYP/def2-SVP implemented in Gaussian16 provided comparable results to that in ORCA 4.2.1.

2.4. GFN2-xTB/MM Metadynamics Simulations

Subsequently, the QM(GFN2-xTB)/MM(CHARMM36) metadynamics method (60) was performed by the collective variables module of NAMD with the NAMD-ORCA interface. (55) One-dimensional potentials of mean force (1D-PMFs) for the enzymatic processes were calculated in the NVT ensemble at 300 K. The C(urea)–O(hydroxide) bond distance, in a range from 1.30 to 3.40 Å, was chosen as the reaction coordinate for nucleophilic attack to form a tetrahedral intermediate. When a proton transfer driven by hydroxide ion was calculated, the reaction coordinate was set to the H(hydroxide)–N(urea) separation ranging from 0.90 to 2.60 Å. For all cases, a hill weight of 0.30 kcal/mol, a hill width of 2.0 Å, and a hill frequency of 50 fs was used. The simulations were performed for 20–30 ps with a 0.5 fs time step of integration.

3. Results and Discussion

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3.1. Possible Mechanisms Starting with 1 and 2

The orientation of urea in 1 before the QM(GFN2-xTB)/MM MD equilibration was suitable for nucleophilic attack of both the terminal and bridging hydroxide ions, with the O···C(urea) separations of 2.37 and 3.40 Å, respectively. The position of H323 was also close to urea consistent with a closed conformation found in KAU, and it would act as a general acid capable of producing leaving ammonia. (14,25) However, the QM(GFN2-xTB)/MM MD equilibration led to a significant structural change. A proton in the proximal NH2 group of urea was spontaneously transferred to the Ni2-bound hydroxide ion, followed by the proton transfer from the doubly protonated H323 to the same amide nitrogen atom. Consequently, the active site complex resulted in having a terminal water ligand W2 and H323 in the neutral form as shown in Figure 2.

Figure 2

Figure 2. QM regions of 1 and 2 after the QM(GFN2-xTB)/MM MD equilibrations (top). Key bond distances are also presented in Å (bottom).

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These trends substantially differed from those in a previous QM study using small active site models (33) in that the activated terminal hydroxide ion spontaneously returned to W2 and thus had no possibility to attack the carbon atom of urea.
The significant structural change found in 1 motivated us to perform an additional calculation using a more accurate broken-symmetry DFT method to judge whether the GFN2-xTB/MM-based equilibration is efficient and reliable. Specifically, the geometry of the QM region in the open-shell singlet state was optimized at the UB3LYP/def2-SV(P) coupled with the RIJCOSX approximation and the def2/J auxiliary basis set. The UB3LYP optimized structure also turned out to be the same as the result of the GFN2-xTB/MM MD equilibration (Figure S4), which reassured us that the activated hydroxide ion coordinated to Ni2 in 1 could act as a base and not a nucleophile regardless of the chosen QM methods. It was also shown that mechanism (iv) was not likely to occur in the presence of protein environment including H323, in agreement with the fact that a cyanate intermediate had never been observed experimentally. By contrast, structural changes such as a bond scission/formation were not observed in 2 during the equilibration (Figure 2).
In this context, urea hydrolysis could proceed through the following two reaction pathways. The first one is the nucleophilic attack of the bridging hydroxide ion on the carbon atom of urea in 1, while W2 acted as a spectator, referred to herein as mechanism (v) (Scheme 2). An analogous mechanism was recently reported by Netto and co-workers. (61) The second one is mechanism (ii) starting with 2.

3.2. Potential Energy Profiles Obtained from QM-Only Cluster Calculations

The differences between the closed-shell singlet and quintet states in the optimized geometries of 1′ and 2′ are comparable. The computed RMSD values for the singlet (quintet) state geometries with respect to the UB3LYP-optimized structures are 0.72 (0.76) and 0.74 (0.68) Å for 1′ and 2′, respectively. In the case of 3′, the RMSD value of 0.28 Å for the quintet state geometry is much smaller than that of 1.08 Å for the singlet state geometry (see also Figure S3). We suggest that the GFN2-xTB calculations in the quintet state are superior to those in the closed-shell singlet state, and thus the quintet-state potential energy surfaces of possible reactions are presented hereafter.
The urea hydrolysis reaction starting with 1′ is found to proceed via mechanism (v) (1R → 1TS1A → 1Int1A → 1TS2A → 1PA) calculated at both the GFN2-xTB and UB3LYP levels (Figure 3a).

Figure 3

Figure 3. Potential energy profiles (in kcal mol–1) and illustrations of the transition state and product structures corresponding to (a) mechanism (v) starting from 1′ and (b) mechanism (ii) starting from 2′ obtained at GFN2-xTB and UB3LYP/def2-SVP levels.

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Cartesian coordinates for all stationary points are presented in the Supporting Information. Figure 3a shows that the nucleophilic attack of the bridging hydroxide on the carbon atom of urea leads to the formation of the O(hydroxide)–C(urea) bond via 1TS1A, and afterward, H(hydroxide) is transferred not directly but via D363 to the NH2 group at 1TS2A. GFN2-xTB predicts that the proton transfer reaction (1TS2A) is the rate-determining step with an activation barrier of 11.5 kcal mol–1. On the other hand, UB3LYP indicates that the first nucleophilic attack (1TS1A) is rate-determining as it is characterized as a late transition state with an O(hydroxide)–C(urea) bond forming distance of 1.55 Å (Table S1) and requires a higher activation barrier than the second step (25.3 vs 23.8 kcal mol –1). The resulting 1PA has a binding mode in line with the active site inhibited by acetohydroxamic acid. (17)
Figure 3b shows that urea hydrolysis starting from 2′ agrees with mechanism (ii). The reaction is initiated by nucleophilic attack of O(hydroxide) on C(urea) via 2TS1A to form a tetrahedral intermediate 2Int1A. The O(hydroxide)···C(urea) distances at 2TS1A are 1.80 and 1.77 Å optimized at the GFN2-xTB and UB3LYP levels (Table S2). GFN2-xTB directly yields an intermediate 2Int3A in which the leaving amide nitrogen atom is triply protonated, while the reaction predicted by UB3LYP passes through an intermediate 2Int2A with the protonated D363 as well as 2Int3A (see also Table S3). The final reaction step is the collapse of 2Int3A involving dissociations of C–N and Ni2–N bonds, leading to 2PA with the same binding mode as 1PA (see also Tables S4 and S5). Unlike mechanism (v), this process is not spontaneous but requires 2TS4A barriers of 11.3 kcal mol–1 for GFN2-xTB and 17.6 kcal mol–1 for UB3LYP.
Based on the QM-only cluster calculations, it appears that mechanism (ii) is preferable for urea hydrolysis on the basis of the potential energy profiles obtained from GFN2-xTB and UB3LYP, though the calculated barrier heights both overestimate the experimental value of 5.7 kcal mol–1. (62,63) Regarding the GFN2-xTB calculations, the highest activation barrier for mechanism (ii) (2TS4A) is slightly lower than that for mechanism (v) (1TS2A), and 2PA is more stable than 1PA. The results of UB3LYP clearly show the superiority of mechanism (ii) in terms of the computed lower barrier heights and larger exothermicity (Figure 3). The dissociation of W2 (1R → 1TSB → 1PB) also turns out much lower activation barriers of 4.9 and 1.5 kcal mol–1 obtained with GFN2-xTB and UB3LYP (Figure S5). This strongly supports that 2′ can be predominantly formed by replacement of W2, as shown in Figure 1. We also find that the GFN2-xTB method can qualitatively reproduce the results of UB3LYP. As such, the use of GFN2-xTB for the QM region can be alternative to broken-symmetry DFT calculations with which simulations on the order of several tens of picoseconds are virtually prohibitive.

3.3. Free Energy Profiles Obtained from GFN2-xTB/MM Metadynamics Calculations

3.3.1. Complex with Monodentate-Bound Urea (1)

Concerning the nucleophilic attack reaction in mechanism (v), the 1D-PMF is depicted in Figure 4 along the C(urea)···O(hydroxide) distance in a range from 1.30 to 3.40 Å. Also shown are the representative snapshots of 1R, 1TS1A, and 1IntA. During the nucleophilic attack, the bridging hydroxide ion and the carboxylic group of D363 keep forming a strong hydrogen-bonding interaction with an O···O distance of 2.82 ± 0.20 Å. It can be seen that 1TS1A for nucleophilic attack has a C–O bond forming distance of ca. 2.0 Å and requires a large activation barrier of 12.7 kcal mol–1. After 1TS1A, the C–O bond length is reduced to ca. 1.4 Å, leading to a tetrahedral intermediate 1IntA lying 6.7 kcal mol–1 above 1R. The high endergonicity of the step is analogous to the QM-only results. It suggests that the monodentate-bound substrate and intermediate at 1TS1A and 1Int1A are not stabilized by intramolecular interactions associated with a closed conformation of the mobile flap. Indeed, the flap of the initial X-ray crystal structure (24) corresponds to the closed conformation in which specific active site residues (H222, H323, and A366) are close to urea, but hydrogen-bonding interactions between urea and these residues are not retained during the simulations. The O(urea)···N(His222), N(urea)···N(H323), and N(urea)···O(A366) distances are 4.21 ± 0.70, 3.42 ± 0.80, and 4.81 ± 0.62 Å, respectively.

Figure 4

Figure 4. (a) One-dimensional potentials of mean force (1D-PMF) of the nucleophilic attack reaction and representative snapshots of the active site in (b) 1R, (c) 1TS1A, and (d) 1IntA, with key distances in Å.

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Subsequently, the reaction undergoes a proton transfer from the bridging hydroxide ion to the leaving NH2 group of urea. Figure 5 shows the free energy profile along the distance between H(hydroxide) and N(urea) atoms, ranging from 0.90 to 2.60 Å. It also indicates that the proton is not transferred directly but via D363 to the NH2 group. The state with protonated D363 is found to be the rate-determining transition state (1TS2A). The computed activation and reaction free energies for 1TS2A and 1PA are +16.4 and −12.4 kcal mol–1 relative to 1IntA. The elimination reaction of the NH2 group is exergonic, possibly because a hydrogen bond is formed in 1PA between the carbamate product and H323, with an N(carbamate)···N(H323) distance of 3.03 ± 0.28 Å. The H222 and A366 residues do not form a stable hydrogen bonding with 1PA as in the case of the nucleophilic attack step. The overall reaction proves to be slightly exergonic by −5.7 kcal mol–1, whereas the proton transfer from the hydroxide ion seems unfavorable on the basis of its high free energy barrier of 23.1 kcal/mol relative to 1R. The activation barrier is also substantially higher than that of the QM-only model (Figure 3), emphasizing that the hydrolysis reaction of urea is more unlikely to proceed in the monodentate complex with the Ni2-coordinated W2.

Figure 5

Figure 5. (a) One-dimensional potentials of mean force (1D-PMF) of the proton transfer reaction and representative snapshots of the active site in (b) 1TS2A and (c) 1PA, with key distances in Å.

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Let us turn our attention to the dissociation of W2. Figure 6 displays its 1D-PMF obtained with the metadynamics simulations and the representative snapshots of the key states denoted as 1R, 1TSB, and 1PB. The Ni2···O(W2) distance ranging from 2.05 to 4.25 Å is used for the reaction coordinate. The activation and reaction free energies are calculated to be 4.5 and 2.0 kcal mol–1 comparable to the QM-only results, and they are considerably lower than those for the aforementioned urea hydrolysis reaction. The dissociated W2 may remain in the vicinity of the active site as suggested by the Ni2···O(W2) separation of ca. 3.5 Å in 1PB. It highlights that, compared with the urea hydrolysis, the W2 dissociation is likely to occur predominantly.

Figure 6

Figure 6. (a) One-dimensional potentials of mean force (1D-PMF) of the W2 dissociation and representative snapshots of the active site in (b) 1TSB and (c) 1PB, with key distances in Å.

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3.3.2. Complex with Bidentate-Bound Urea (2)

The 1D-PMF corresponding to the nucleophilic attack is depicted along the C(urea)···O(hydroxide) distance in a range from 1.30 to 3.40 Å (Figure 7). The representative snapshots of 2R, 2TS1A, and 2IntA are also shown in Figure 7. Inspection of 2R reveals that urea can first bind to the dinickel center in a monodentate mode even in the absence of W2, having a longer Ni2···N(urea) separation of ca. 3.3 Å. In line with 1, a strong hydrogen bonding is formed between the bridging hydroxide ion and the carboxylic group of D363, with an O···O distance of 2.74 ± 0.18 Å.

Figure 7

Figure 7. (a) One-dimensional potentials of mean force (1D-PMF) of the nucleophilic attack reaction and representative snapshots of the active site in (b) 2R, (c) 2TS1A, and (d) 2IntA, with key distances in Å.

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The nucleophilic attack step not only shortens the C(urea)–O(hydroxide) bond length but also increases the binding of one of the NH2 group of urea to Ni2. The C–O bond forming distance of ca. 2.0 Å at 2TS1A is similar to that at 1TS1A. The Ni2–N(urea) bond lengths at 2TS1A and 2IntA are reduced to 2.47 and 2.30 Å, respectively (Figure 7). The resulting activation free energy of 4.0 kcal mol–1 is close to experimentally measured values of ca. 5.7 kcal mol–1, (62,63) and the reaction free energy of −4.5 kcal mol–1 is obviously lower compared with the result for mechanism (v) discussed above (6.7 kcal mol–1). These values are much lower than the corresponding values of +10.6 and −2.4 kcal mol–1 obtained from the QM-only model (Figure 3b). As such, the Ni2–N(urea) bond formation that occurs concomitantly with the nucleophilic attack can stabilize 2TS1A and 2IntA, causing a change of the binding mode from monodentate to bidentate. We also find that the bidentate complexes at 2TS1A and 2IntA form multiple hydrogen bonds with H222 as well as with A366 in which either or both NH2 groups are involved. The O(urea)···N(His222), the proximal N(urea)···O(A366), and the distal N(urea)···O(A366) distances are 3.01 ± 0.27, 3.17 ± 0.30, and 3.30 ± 0.31 Å, respectively. These features are consistent with the previous hypothesis (64) that closure of the flap may be responsible for the coordination of the urea NH2 group to Ni2 and the formation of multiple hydrogen bonds with the nearby residues. In addition, it should be underlined that the active site can gain the benefits of the closed conformation only after replacement of W2, affording a coordinatively unsaturated Ni2 site.
Upon the formation of 2IntA, a proton transfer occurs from the bridging hydroxide ion to the leaving NH2 group only via 2TS2A, while the QM-only model in the absence of the protein environment involves 2Int3A and 2TS4A. Figure 8 shows the free energy profile along the distance between H(hydroxide) and N(urea) atoms in a range from 0.90 to 2.60 Å. Unlike the free energy landscape for mechanism (v) illustrated in Figure 5, the proton transfer step of mechanism (ii) turns out to be highly exergonic with 2TS2A and 2PA having free energies of −1.7 and −22.5 kcal mol–1 relative to 2R. In 2PA, the formed carbamate is coordinated to the metal center in a bidentate manner consistent with a urease complexed with diammonium phosphate. (23) We see that the rigidity of the bidentate-bound tetrahedral intermediate and carbamate can maintain a hydrogen bond with H222 in the formation of 2PA continuing on the nucleophilic attack reaction, as the distance between the carbonyl oxygen atom and N(His222) decreases to 2.91 ± 0.18 Å. The generated NH3 also forms a weak hydrogen bond with D363 based on an N(NH3)···O(D363) distance of 3.06 ± 0.35 Å, whereas A366 and H323 seem not to play a significant role. Overall, the formation of the bidentate complex is crucial for catalysis because the subsequent reactions can proceed rapidly with benefits of the stabilization provided by the metal center and the surrounding protein.

Figure 8

Figure 8. (a) One-dimensional potentials of mean force (1D-PMF) of the proton transfer reaction and representative snapshots of the active site in (b) 2TS2A and (c) 2PA, with key distances in Å.

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4. Conclusions

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The present study investigates the relationship between substrate binding and the catalytic mechanism of urease and explores possible reaction mechanisms with the use of QM-only cluster calculations and QM/MM free energy simulations. The QM region including the dinickel center and certain active site residues is treated by GFN2-xTB, which shows good performance in reproducing structures of both coordinatively unsaturated and saturated active-site model complexes. The QM(GFN2-xTB)/MM(CHARMM36) simulations provide evidence that the bridging hydroxide ion acts as both a nucleophile and a general acid, whereas a hydroxide ion that arises from the deprotonation of the Ni2-bound W2 can be excluded from a candidate for a nucleophile. The metadynamics simulations clearly demonstrate that urea hydrolysis via an active site complex without W2 (2) is much more plausible than via that with W2 (1), judging from the computed activation free energies for the rate-limiting step (4.0 vs 23.1 kcal mol–1) compared with the experimentally measured data (5.7 kcal mol–1). The high activation barriers for the hydrolysis process starting from 2 reflect the lack of stabilization via hydrogen-bonding interactions with nearby residues. We also reveal that the energetically favorable dissociation of W2 is a key step that triggers the binding of urea in an appropriate position prior to the hydrolysis process. The subsequent nucleophilic attack reaction involves a switch of the binding mode from monodentate to bidentate. In addition to the formation of the bidentate complex, comparison between the QM-only and QM/MM MD results demonstrates the importance of including the protein environment for the successive decomposition process. Indeed, the proton transfer reactions via D363 are significantly stabilized by forming multiple hydrogen bond interactions with the active site residues. Further studies are needed on the difference in reaction mechanism between urea and candidates for inhibitors to design more potent inhibitors. This work is in progress.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.1c10200.

  • Figure S1, active site models for 1 and 2 excluding H222, H323, and A366 with key distances and spin density; Cartesian coordinates of these models obtained with the UB3LYP/def2-SVP level; the created topology and parameter files; Figure S2, RMSDs with respect to the initial conformations during a 10 ns classical MD simulation for 1 and 2; Figure S3, overlay of smaller models (1, 2, and 3) optimized in the singlet and quintet states at GFN2-xTB with respect to the structure obtained with the broken-symmetry DFT method; Figure S4, structure and Cartesian coordinates of the QM region for 1 optimized in the open-shell singlet state at the UB3LYP/def2-SV(P) level; Figure S5, potential energy profile of the dissociation of W2; Cartesian coordinates for all stationary points of the QM-only cluster calculations optimized at the GFN2-xTB and UB3LYP levels; Tables S1–S5, key bond distance for all stationary points obtained from QM-only cluster and QM/MM metadynamics calculations; full citation for ref (59) (PDF)

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Author Information

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  • Corresponding Author
  • Author
    • Yu Takano - Department of Biomedical Information Sciences, Graduate School of Information Sciences, Hiroshima City University, 3-4-1 Ozuka-Higashi, Asa-Minami-Ku, Hiroshima 731-3194 Japan;  Orcidhttps://orcid.org/0000-0002-4165-8672
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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T.S. acknowledges the fund for the Promotion of Joint International Research (Fostering Joint International Research (B)) and for the Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (Nos. 18KK0194 and 21K04985). Y.T acknowledges the fund for the Grants-in-Aid for Scientific Research (C) from the JSPS (No. 19K06589). Y.T. is also grateful to the Ministry of Education, Culture, Sports, Science and Technology (MEXT) for a Grant-in-Aid for Scientific Research on Innovative Areas “3D active-site science” (No. 26105012) and Transformative Research Areas (A) “Progressive condensed matter physics inspired by hyper-ordered structures” (No. 20H05883). Some computations were performed using Research Center for Computational Science, Okazaki, Japan.

References

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This article references 64 other publications.

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    Abstr.: This review is an attempt to retrace the chronicle that starts from the discovery of the role of nickel as the essential metal ion in urease for the enzymic catalysis of urea, a key step in the biogeochem. cycle of nitrogen on Earth, to the most recent progress in understanding the chem. of this historical enzyme. Data and facts are presented through the magnifying lenses of the authors, using their best judgment to filter and elaborate on the many facets of the research carried out on this metalloenzyme over the years. The tale is divided in chapters that discuss and describe the results obtained in the subsequent leaps in the knowledge that led from the discovery of a biol. role for Ni to the most recent advancements in the comprehension of the relationship between the structure and function of urease. This review is intended not only to focus on the bioinorg. chem. of this beautiful metal-based catalysis, but also, and maybe primarily, to evoke inspiration and motivation to further explore the realm of bio-based coordination chem.
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    Covering: up to the end of 2017The human body is composed of an equal no. of human and microbial cells. While the microbial community inhabiting the human gastrointestinal tract plays an essential role in host health, these organisms have also been connected to various diseases. Yet, the gut microbial functions that modulate host biol. are not well established. In this review, we describe metabolic functions of the human gut microbiota that involve metalloenzymes. These activities enable gut microbial colonization, mediate interactions with the host, and impact human health and disease. We highlight cases in which enzyme characterization has advanced our understanding of the gut microbiota and examples that illustrate the diverse ways in which metalloenzymes facilitate both essential and unique functions of this community. Finally, we analyze Human Microbiome Project sequencing datasets to assess the distribution of a prominent family of metalloenzymes in human-assocd. microbial communities, guiding future enzyme characterization efforts.
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    Bremner, J. M.; Krogmeier, M. J. Evidence that the Adverse Effect of Urea Fertilizer on Seed Germination in Soil is due to Ammonia Formed through Hydrolysis of Urea by Soil Urease. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 8185– 8188,  DOI: 10.1073/pnas.86.21.8185
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    Studies using seeds of wheat (Triticum aestivum), rye (Secale cereale), barley (Hordeum vulgare) and corn (Zea mays) indicated that the adverse effect of urea fertilizer on seed germination in soil is due to ammonia formed through hydrolysis of urea by soil urease and is not due to urea itself, to urea fertilizer impurities such as biuret, or to nitrite formed by nitrification of urea nitrogen. Support for this conclusion was obtained from (i) comparison of the effects on seed germination in soil of purified urea, urea fertilizers, urea fertilizer impurities, and compds. formed by enzymic and microbial transformations of urea in soil; (ii) studies showing that ammonia volatilized from soils treated with urea completely inhibited germination of seeds close to, but not in contact with, these soils; and (iii) expts. showing that the adverse effect of urea fertilizer on seed germination in soil was completely eliminated when the soil was autoclaved to destroy urease or was treated with phenylphosphorodiamidate to inhibit soil urease activity before treatment with urea fertilizer.
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    Gioacchini, P.; Nastri, A.; Marzadori, C.; Giovannini, C.; Antisari, L. V.; Gessa, C. Influence of Urease and Nitrification Inhibitors on N Losses from Soils Fertilized with Urea. Biol. Fertil. 2002, 36, 129– 135,  DOI: 10.1007/s00374-002-0521-1
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    The aim was to evaluate how the N losses through volatilization and leaching from soils fertilized with urea can be affected by the application of a urease inhibitor or a urease plus a nitrification inhibitor. The expt. was carried out using lysimeters with 15N-labeled urea and N-butylthiophosphoric triamide (NBPT) as urease inhibitor and dicyandiamide (DCD) as nitrification inhibitor, comparing 3 treatments: urea alone (U), urea + NBPT (UN) and urea + NBPT + DCD (UND). Both volatilization and leaching were significantly different in the soils used, according to their physicochem. characteristics. However, the pattern of the loss was similar: the volatilization was significantly reduced by NBPT (UN), but the presence of DCD (UND) significantly increased the loss, with respect to UN. Considering leaching, the highest amt. of NO3- was lost with UND, the lowest with U. The greatest amt. of N lost by leaching was soil-derived N produced by the N mineralization-immobilization turnover. We suggest that, by maintaining the NH4+ in the soils, the inhibitors, in particular DCD, caused a priming effect with a subsequent increase in the rate of soil org. matter mineralization and an extra release of soil org. N. The priming effect was real in the sandy loam (SL) soil where a net N release was obsd., whereas in the clay loam (CL) soil the effect of the inhibitors was less pronounced and an apparent priming effect was obsd.; however, a real priming effect also cannot be excluded in this soil.
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    H. pylori, an etiol. agent in a variety of gastro-duodenal diseases, produces a large amt. of urease (I), which is believed to neutralize gastric acid by producing NH3 for the survival of the bacteria. Up to 30% of I assocs. with the surface of intact cells upon lysis of neighboring bacteria. The role of I at the extracellular location has been a subject of controversy because the purified enzyme is irreversibly inactivated below pH 5. Here, the authors detd. the crystal structure of H. pylori I, which had a 1.1 MDa spherical assembly of 12 catalytic units with an outer diam. of ∼160 Å. Under physiol. relevant conditions, the activity of the enzyme remains unaffected down to pH 3. Activity assays under different conditions indicated that the duster of the 12 active sites on the supramol. assembly may be crit. for the survival of the enzyme at low pH. The structure provides a novel example of a mol. assembly adapted for acid resistance that, together with the low Km of the I, is likely to enable the organism to inhabit the hostile niche.
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    Phillips, K.; Munster, D. J.; Allardyce, R. A.; Bagshaw, P. F. Antibacterial Action of the Urease Inhibitor Acetohydroxamic acid on Helicobacter pylori. J. Clin. Pathol. 1993, 46, 372– 373,  DOI: 10.1136/jcp.46.4.372
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    The urease inhibitor acetohydroxamic acid (AHA) was assessed for its bacteriostatic and bactericidal effects on Helicobacter pylori. For eight isolates of H pylori, the minimum inhibitory concentration (MIC) was either 200 mg/l or 400 mg/l. Interactions between AHA and antimicrobial drugs used to treat H pylori were also determined. For most isolates AHA reduced the MIC for colloidal bismuth subcitrate (CBS), tetracycline, metronidazole, and amoxicillin. In a few isolates, however, AHA increased the minimum bactericidal concentration (MBC) for these antimicrobial treatments. In vitro AHA is active against H pylori and it interacts with other agents directed against H pylori.
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    Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Miletti, S.; Ciurli, S.; Mangani, S. The Complex of Bacillus pasteurii Urease with Acetohydroxamate Anion from X-ray Data at 1.55 Å Resolution. J. Biol. Inorg. Chem. 2000, 5, 110– 118,  DOI: 10.1007/s007750050014
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    The complex of Bacillus pasteurii urease with acetohydroxamate anion from x-ray data at 1.55 Å resolution
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    JBIC, Journal of Biological Inorganic Chemistry (2000), 5 (1), 110-118CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)
    The crystal structure of B. pasteurii urease inhibited by acetohydroxamic acid was solved and refined anisotropically using synchrotron x-ray cryogenic diffraction data (1.55 Å resoln.; 99.5% completeness; data redundancy = 26; R-factor = 15.1%, PDB code 4UBP). The 2 Ni2+ ions in the active site were sepd. by a distance of 3.53 Å. The structure clearly showed the binding mode of the inhibitor anion, sym. bridging the 2 Ni2+ ions in the active site through the hydroxamate O atom and chelating 1 Ni2+ ion through the carbonyl O atom. The flexible flap flanking the active site cavity was in the open conformation. The possible implications of the results on structure-based mol. design of new urease inhibitors were discussed.
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    Benini, S.; Rypniewski, W.; Wilson, K.; Ciurli, S.; Mangani, S. Structure-based Rationalization of Urease Inhibition by Phosphate: Novel Insights into the Enzyme Mechanism. J. Biol. Inorg. Chem. 2001, 6, 778– 790,  DOI: 10.1007/s007750100254
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    Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Ciurli, S.; Mangani, S.
    JBIC, Journal of Biological Inorganic Chemistry (2001), 6 (8), 778-790CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)
    The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron x-ray diffraction data from a vitrified crystal (1.85 Å resoln., 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 Å separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom sym. bridges the two metal ions (1.9-2.0 Å), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 Å. The fourth phosphate oxygen is directed into the active site channel. Anal. of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4- anion, and that an addnl. proton is present on the Oδ2 atom of Aspα363, an active site residue involved in Ni coordination through Oδ1. The flexible flap flanking the active site cavity is in the open conformation. Anal. of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.
  20. 20
    Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Mangani, S.; Ciurli, S. Molecular Details of Urease Inhibition by Boric Acid: Insights into the Catalytic Mechanism. J. Am. Chem. Soc. 2004, 126, 3714– 3715,  DOI: 10.1021/ja049618p
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    Molecular Details of Urease Inhibition by Boric Acid: Insights into the Catalytic Mechanism
    Benini, Stefano; Rypniewski, Wojciech R.; Wilson, Keith S.; Mangani, Stefano; Ciurli, Stefano
    Journal of the American Chemical Society (2004), 126 (12), 3714-3715CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The structure of the complex of urease, a Ni-contg. metalloenzyme, with boric acid was detd. at 2.10 Å resoln. The complex shows the unprecedented binding mode of the competitive inhibitor to the dinuclear metal center, with the B(OH)3 mol. bridging the Ni ions and leaving in place the bridging hydroxide. Boric acid can be considered a substrate analog of urea, and the structure supports the proposal that the Ni-bridging hydroxide acts as the nucleophile in the enzymic process of urea hydrolysis.
  21. 21
    Benini, S.; Cianci, M.; Mazzei, L.; Ciurli, S. Fluoride inhibition of Sporosarcina pasteurii urease: structure and thermodynamics. J. Biol. Inorg. Chem. 2014, 19, 1243– 1261,  DOI: 10.1007/s00775-014-1182-x
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    Fluoride inhibition of Sporosarcina pasteurii urease: structure and thermodynamics
    Benini, Stefano; Cianci, Michele; Mazzei, Luca; Ciurli, Stefano
    JBIC, Journal of Biological Inorganic Chemistry (2014), 19 (8), 1243-1261CODEN: JJBCFA; ISSN:0949-8257. (Springer)
    Urease is a Ni-dependent enzyme and a virulence factor for ureolytic bacterial human pathogens, but it is also necessary to convert urea, the most worldwide-used fertilizer, into forms of nitrogen that can be taken up by crop plants. A strategy to control the activity of urease for medical and agricultural applications is to use enzyme inhibitors. F- is a known urease inhibitor, but the structural basis of its mode of inhibition is still undetd. Here, kinetic studies on the F--induced inhibition of urease from S. pasteurii, a widespread and highly ureolytic soil bacterium, were performed using isothermal titrn. calorimetry and revealed a mixed competitive and uncompetitive mechanism. The pH dependence of the Ki values, investigated in the pH 6.5-8.0 range, revealed a predominant uncompetitive mechanism that increased by increasing the pH, and a lesser competitive inhibition that increased by lowering the pH. Ten crystal structures of the enzyme were independently detd. using 5 crystals of the native form and 5 crystals of the protein crystd. in the presence of F-. The anal. of these structures revealed the presence of 2 F- anions coordinated to the Ni2+ ions in the active site, in terminal and bridging positions. The present study consistently supported an interaction of F- with the Ni centers in the urease active site in which one F- anion competitively bound to Ni2+ proposed to coordinate urea in the initial step of the catalytic mechanism, while another F- anion uncompetitively substituted the Ni2+-bridging hydroxide, blocking its nucleophilic attack on urea.
  22. 22
    Mazzei, L.; Cianci, M.; Musiani, F.; Lente, G.; Palombo, M.; Ciurli, S. Inactivation of Urease by Catechol: Kinetics and Structure. J. inorg. Biochem. 2017, 166, 182– 189,  DOI: 10.1016/j.jinorgbio.2016.11.016
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    22
    Inactivation of urease by catechol: Kinetics and structure
    Mazzei, Luca; Cianci, Michele; Musiani, Francesco; Lente, Gabor; Palombo, Marta; Ciurli, Stefano
    Journal of Inorganic Biochemistry (2017), 166 (), 182-189CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
    Urease is a Ni(II)-contg. enzyme that catalyzes the hydrolysis of urea to yield NH3 and carbamate at a rate 1015-fold higher than the uncatalyzed reaction. Urease is a virulence factor of several human pathogens, in addn. to decreasing the efficiency of soil org. nitrogen fertilization. Therefore, efficient urease inhibitors are actively sought. Here, the authors describe the characterization of the interaction between urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) with catechol, a model polyphenol. In particular, catechol irreversibly inactivated both SPU and JBU with a complex radical-based autocatalytic multistep mechanism. The crystal structure of the SPU-catechol complex, detd. at 1.50 Å resoln., revealed the structural details of the enzyme inhibition.
  23. 23
    Mazzei, L.; Cianci, M.; Contaldo, U.; Ciurli, S. Insights into Urease Inhibition by N-(n-Butyl) Phosphoric Triamide through an Integrated Structural and Kinetic Approach. J. Agric. Food Chem. 2019, 67, 2127– 2138,  DOI: 10.1021/acs.jafc.8b04791
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    23
    Insights into urease inhibition by N-(n-butyl) phosphoric triamide through an integrated structural and kinetic approach
    Mazzei, Luca; Cianci, Michele; Contaldo, Umberto; Ciurli, Stefano
    Journal of Agricultural and Food Chemistry (2019), 67 (8), 2127-2138CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    The nickel-dependent enzyme urease represents a neg. element for the efficiency of soil nitrogen fertilization as well as a virulence factor for a large no. of pathogenic and antibiotic-resistant bacteria. The development of ever more efficient urease inhibitors demands knowledge of their modes of action at the mol. level. N-(n-Butyl)-phosphoric triamide (NBPTO) is the oxo-deriv. of N-(n-butyl)-thiophosphoric triamide (NBPT), which is extensively employed in agriculture to increase the efficiency of urea-based fertilizers. The 1.45 Å resoln. structure of the enzyme-inhibitor complex obtained upon incubation of Sporosarcina pasteurii urease (SPU) with NBPTO shows the presence of diamido phosphoric acid (DAP), generated upon enzymic hydrolysis of NBPTO with the release of Bu amine. DAP is bound in a tridentate binding mode to the two Ni(II) ions in the active site of urease via two O atoms and an amide NH2 group, whereas the second amide group of DAP points away from the metal center into the active-site channel. The mobile flap modulating the size of the active-site cavity is found in a disordered closed-open conformation. A kinetic characterization of the NBPTO-based inhibition of both bacterial (SPU) and plant (Canavalia ensiformis or jack bean, JBU) ureases, carried out by calorimetric measurements, indicates the occurrence of a reversible slow-inhibition mode of action. The latter is characterized by a very small value of the equil. dissocn. const. of the urease-DAP complex caused, in turn, by the large rate const. for the formation of the enzyme-inhibitor complex. The much greater capability of NBPTO to inhibit urease, as compared with that of NBPT, is thus not caused by the presence of a P=O moiety vs. a P=S moiety, as previously suggested, but rather by the readiness of NBPTO to react with urease without the need to convert one of the P-NH2 amide moieties to its P-OH acid deriv., as in the case of NBPT. The latter process is indeed characterized by a very small equil. const. that reduces drastically the concn. of the active form of the inhibitor in the case of NBPT. This indicates that high-efficiency phosphoramide-based urease inhibitors must have at least one O atom bound to the central P atom in order for the mol. to efficiently and rapidly bind to the dinickel center of the enzyme.
  24. 24
    Mazzei, L.; Cianci, M.; Benini, S.; Ciurli, S. The Structure of the Elusive Urease-Urea Complex Unveils the Mechanism of a Paradigmatic Nickel-Dependent Enzyme. Angew. Chem., Int. Ed. 2019, 58, 7415– 7419,  DOI: 10.1002/anie.201903565
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    24
    The structure of the elusive urease-urea complex unveils the mechanism of a paradigmatic nickel-dependent enzyme
    Mazzei, Luca; Cianci, Michele; Benini, Stefano; Ciurli, Stefano
    Angewandte Chemie, International Edition (2019), 58 (22), 7415-7419CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Urease, the most efficient enzyme known, contains an essential dinuclear NiII cluster in the active site. It catalyzes the hydrolysis of urea, inducing a rapid pH increase that has neg. effects on human health and agriculture. Thus, the control of urease activity is of utmost importance in medical, pharmaceutical, and agro-environmental applications. All known urease inhibitors are either toxic or inefficient. The development of new and efficient chems. able to inhibit urease relies on the knowledge of all steps of the catalytic mechanism. The short (microseconds) lifetime of the urease-urea complex has hampered the detn. of its structure. The present study uses fluoride to substitute the hydroxide acting as the co-substrate in the reaction, preventing the occurrence of the catalytic steps that follow substrate binding. The 1.42 Å crystal structure of the urease-urea complex, reported here, resolves the enduring debate on the mechanism of this metalloenzyme.
  25. 25
    Karplus, P. A.; Pearson, M. A.; Hausinger, R. P. 70 Years of Crystalline Urease: What Have We Learned?. Acc. Chem. Res. 1997, 30, 330– 337,  DOI: 10.1021/ar960022j
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    70 Years of crystalline urease: What have we learned?
    Karplus, P. Andrew; Pearson, Matthew; Hausinger, Robert P.
    Accounts of Chemical Research (1997), 30 (8), 330-337CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review with 60 refs. on the functional and structural characteristics of Klebsiella aerogenes urease. Crystallog. and functional studies of wild-type urease and some site-directed variants suggest a detailed catalytic mechanism which accounts for its kinetic properties and its structure. This mechanism assigns the limiting pKa's of 6.4 and 9.0 to the general acid His-320 and the hydrolytic water, resp. This assignment is such that only a small fraction of the urease mols. are present in the correct protonation state for activity. Obsd. disorder of the solvation in the active site and the comparison of multiple structures provide evidence for suboptimal interactions in the water-filled active site. These suboptimal interactions may lead to an enthalpy-driven free energy gain when this water is released into the bulk solvent upon urea binding. As such, the relaxation of the high-energy state could provide a large amt. of binding energy required for catalysis.
  26. 26
    Pearson, M. A.; Park, I.-S.; Schaller, R. A.; Michel, L. O.; Karplus, P. A.; Hausinger, R. P. Kinetic and Structural Characterization of Urease Active Site Variants. Biochemistry 2000, 39, 8575– 8584,  DOI: 10.1021/bi000613o
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    26
    Kinetic and Structural Characterization of Urease Active Site Variants
    Pearson, Matthew A.; Park, Il-Seon; Schaller, Ruth A.; Michel, Linda O.; Karplus, P. Andrew; Hausinger, Robert P.
    Biochemistry (2000), 39 (29), 8575-8584CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >1014-fold the spontaneous rate. To better define the enzyme mechanism, we examd. the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar ∼10-5-fold deficiencies in rates, modest Km changes, and disorders in the peptide flap covering their active sites. H219A urease exhibits 103-fold increased Km over that of native enzyme, whereas the increase is less marked (∼102-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water mols. covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity (∼10-3 that of native enzyme), a small increase in Km, and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a ∼10-4-fold decreased catalytic rate with near-normal pH dependence and an unaffected Km. Phenylglyoxal inactivates the R336Q variant at over half the rate obsd. for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of kcat and kcat/Km for the native enzyme masks a more complex underlying pH dependence involving at least four pKas.
  27. 27
    Dixon, N. E.; Blakeley, R. L.; Zerner, B. Jack Bean Urease (EC 3.5.1.5). III. The Involvement of Active-site Nickel Ion in Inhibition by b-Mercaptoethanol, Phosphoramidate, and Fluoride. Can. J. Biochem. 1980, 58, 481– 488,  DOI: 10.1139/o80-064
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    27
    Jack bean urease (EC 3.5.1.5). III. The involvement of active-site nickel ion in inhibition by β-mercaptoethanol, phosphoramidate, and fluoride
    Dixon, Nicholas E.; Blakeley, Robert L.; Zerner, Burt
    Canadian Journal of Biochemistry (1980), 58 (6), 481-8CODEN: CJBIAE; ISSN:0008-4018.
    Interaction of β-mercaptoethanol with urease produces large, rapid, and fully reversible spectral changes in that part of the electronic absorption spectrum which is assocd. with the tightly bound Ni2+. The spectrophotometrically detd. value of the dissocn. const. of the β-mercaptoethanol-urease complex (0.95 mM at pH 7.12 and 25°) is in agreement with the Ki (0.72 mM) for β-mercaptoethanol acting as a competitive inhibitor in the hydrolysis of urea. This constitutes direct evidence that the Ni2+ in jack bean urease is at the active site. Inhibition of urease by phosphoramidate is slowly achieved and slowly reversed, and upon reactivation of the isolated phosphoramidate-urease complex, phosphoramidate is regenerated in good yield. Spectrophotometric expts. indicate that phosphoramidate binds to Ni2+ in urease. Competition with β-mercaptoethanol was used to det. a dissocn. const. (1.23 mM at pH 7.12 and 25°) for a F--urease complex in which F- also coordinates with an active-site Ni2+. Kinetic evidence is presented which indicates that in the presence of urea, a ternary complex (F-urea-urease) is formed.
  28. 28
    Pelmenschikov, V.; Siegbahn, P. E. Nickel Superoxide Dismutase Reaction Mechanism Studied by Hybrid Density Functional Methods. J. Am. Chem. Soc. 2006, 128, 7466– 7475,  DOI: 10.1021/ja053665f
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    Nickel Superoxide Dismutase Reaction Mechanism Studied by Hybrid Density Functional Methods
    Pelmenschikov, Vladimir; Siegbahn, Per E. M.
    Journal of the American Chemical Society (2006), 128 (23), 7466-7475CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The reaction mechanism for the disproportionation of the toxic superoxide radical to mol. oxygen and hydrogen peroxide by the nickel-dependent superoxide dismutase (NiSOD) has been studied using the B3LYP hybrid DFT method. Based on the recent x-ray structures of the enzyme in the resting oxidized Ni(III) and x-ray-reduced Ni(II) states, the model investigated includes the backbone spacer of six residues (sequence nos. 1-6) as a structural framework. The side chains of residues His-1, Cys-2, and Cys-6, which are essential for nickel binding and catalysis, were modeled explicitly. The catalytic cycle consists of two half-reactions, each initiated by the successive substrate approach to the metal center. The two protons necessary for the dismutation are postulated to be delivered concertedly with the superoxide radical anions. The first (reductive) phase involves Ni(III) redn. to Ni(II), and the second (oxidative) phase involves the metal reoxidn. back to its resting state. The Cys-2 thiolate sulfur serves as a transient protonation site in the interim between the two half-reactions, allowing for the dioxygen and hydrogen peroxide mols. to be released in the reductive and oxidative phases, resp. The His-1 side chain nitrogen and backbone amides of the active site channel are shown to be less favorable transient proton locations, as compared to the Cys-2 sulfur. Comparisons are made to the Cu- and Zn-dependent SOD, studied previously using similar models.
  29. 29
    Sparta, M.; Valdez, C. E.; Alexandrova, A. N. Metal-dependent Activity of Fe and Ni Acireductone Dioxygenases: How Two Electrons Reroute the Catalytic Pathway. J. Mol. Biol. 2013, 425, 3007– 3018,  DOI: 10.1016/j.jmb.2013.05.001
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    Metal-Dependent Activity of Fe and Ni Acireductone Dioxygenases: How Two Electrons Reroute the Catalytic Pathway
    Sparta, Manuel; Valdez, Crystal E.; Alexandrova, Anastassia N.
    Journal of Molecular Biology (2013), 425 (16), 3007-3018CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Two virtually identical acireductone dioxygenases, ARD and ARD', catalyze completely different oxidn. reactions of the same substrate, 1,2-dihydroxy-3-keto-5-(methylthio)pentene, depending exclusively on the nature of the bound metal. Fe2+-dependent ARD' produces the α-keto acid precursor of methionine and formate and allows for the recycling of methionine in cells. Ni2+-dependent ARD instead produces methylthiopropionate, CO, and formate, and exits the methionine salvage cycle. This mechanistic difference has not been understood to date but has been speculated to be due to the difference in coordination of the substrate to Fe2+ vs. Ni2+: forming a five-membered ring vs. a six-membered ring, resp., thus exposing different carbon atoms for the attack by O2. Here, using mixed quantum-classical mol. dynamics simulations followed by the d. functional theory mechanistic investigation, we show that contrary to the old hypothesis, both metals preferentially bind the substrate as a six-membered ring, exposing the exact same sites to the attack by O2. It is the electronic properties of the metals that are then responsible for the system following different reaction paths, to yield the resp. products. We fully explain the puzzling metal-induced difference in functionality between ARD and ARD' and, in particular, propose a new mechanism for ARD'. All results are in agreement with available isotopic substitution and other exptl. data.
  30. 30
    Wang, W.-J.; Wei, W.-J.; Liao, R.-Z. Deciphering the Chemoselectivity of Nickel-dependent Quercetin 2, 4-Dioxygenase. Phys. Chem. Chem. Phys. 2018, 20, 15784– 15794,  DOI: 10.1039/C8CP02683A
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    Deciphering the chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase
    Wang, Wen-Juan; Wei, Wen-Jie; Liao, Rong-Zhen
    Physical Chemistry Chemical Physics (2018), 20 (23), 15784-15794CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
    The reaction mechanism and chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase (2,4-QueD) have been investigated using the QM/MM approach. The protonation state of the Glu74 residue, a first-shell ligand of Ni, has been considered to be either neutral or deprotonated. QM/MM calcns. predict that Glu74 must be deprotonated to rationalize the chemoselectivity and steer the 2,4-dioxygenolytic cleavage of quercetin, which harvests the exptl.-obsd. product, 2-protocatechuoylphloroglucinol carboxylic acid, coupled with the release of carbon monoxide. If the enzyme has a neutral Glu74 residue, the undesired 2,3-dioxygenolytic cleavage of quercetin becomes the dominant pathway, leading to the formation of α-keto acid. The calcns. suggest that the reaction takes place via three major steps: (1) attack of the superoxide on the C2 of the substrate pyrone ring to generate a NiII-peroxide intermediate; (2) formation of the second C-O bond between C4 and the peroxide to produce a peroxide bridge; (3) simultaneous cleavage of the C2-C3, C3-C4, and O1-O2 bonds with the formation of 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The third step was found to be rate-limiting, with a barrier of 17.4 kcal mol-1, which is in very good agreement with the exptl. kinetic data. For the second C-O bond formation, an alternative pathway is that the peroxide attacks the C3 of the substrate pyrone ring, leading to the formation of a four-membered ring intermediate, which then undergoes concerted C2-C3 and O1-O2 bond cleavages to produce an α-keto acid. This pathway is assocd. with a barrier of 30.6 kcal mol-1, which is much higher than the major pathway. When Glu74 is protonated, the 2,3-dioxygenolytic pathway, however, has a lower barrier (21.8 kcal mol-1) than the 2,4-dioxygenolytic pathway.
  31. 31
    Siegbahn, P. E.; Chen, S.-L.; Liao, R.-Z. Theoretical Studies of Nickel-dependent Enzymes. Inorganics 2019, 7, 95,  DOI: 10.3390/inorganics7080095
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    Theoretical studies of nickel-dependent enzymes
    Siegbahn, Per E. M.; Chen, Shi-Lu; Liao, Rong-Zhen
    Inorganics (2019), 7 (8), 95CODEN: INORCW; ISSN:2304-6740. (MDPI AG)
    The advancements of quantum chem. methods and computer power allow detailed mechanistic investigations of metalloenzymes. In particular, both quantum chem. cluster and combined QM/MM approaches have been used, which have been proven to successfully complement exptl. studies. This review starts with a brief introduction of nickel-dependent enzymes and then summarizes theor. studies on the reaction mechanisms of these enzymes, including NiFe hydrogenase, methyl-coenzyme M reductase, nickel CO dehydrogenase, acetyl CoA synthase, acireductone dioxygenase, quercetin 2,4-dioxygenase, urease, lactate racemase, and superoxide dismutase.
  32. 32
    Suárez, D.; Díaz, N.; Merz, K. M. Ureases: Quantum Chemical Calculations on Cluster Models. J. Am. Chem. Soc. 2003, 125, 15324– 15337,  DOI: 10.1021/ja030145g
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    Ureases: Quantum Chemical Calculations on Cluster Models
    Suarez, Dimas; Diaz, Natalia; Merz, Kenneth M., Jr.
    Journal of the American Chemical Society (2003), 125 (50), 15324-15337CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Herein, the authors present results from a computational study of dinickel complexes that are relevant to the catalytic hydrolysis of urea exerted by the urease enzymes. The B3LYP d. functional is used to characterize the equil. geometry, electronic and magnetic properties, and energies for a series of realistic complexes modeling the active site of ureases. The anal. of the theor. results gives new insight into the structure, substrate binding, and catalytic mechanism. The water bridge between the two Ni(II) ions obsd. in the crystallog. structures of the ureases was assigned to a hydroxide bridge in agreement with the obsd. small antiferromagnetic coupling. Both monodentate and bidentate urea-bound complexes, in which urea had favorable orientations for catalysis, were characterized. Finally, two reaction mechanisms were investigated starting from the monodentate and bidentate urea-bound complexes, resp. Both a Ni1···Ni2 bridging hydroxide and a Ni2-bound water mol. play crucial roles in the two mechanisms.
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    Estiu, G.; Merz, K. M. Competitive Hydrolytic and Elimination Mechanisms in the Urease Catalyzed Decomposition of Urea. J. Phys. Chem. B 2007, 111, 10263– 10274,  DOI: 10.1021/jp072323o
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    Competitive Hydrolytic and Elimination Mechanisms in the Urease Catalyzed Decomposition of Urea
    Estiu, Guillermina; Merz, Kenneth M., Jr.
    Journal of Physical Chemistry B (2007), 111 (34), 10263-10274CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    We present a high-level quantum chem. study of possible elimination reaction mechanisms assocd. with the catalytic decompn. of urea at the binuclear nickel active site cluster of urease. Stable intermediates and transition state structures have been identified along several possible reaction pathways. The computed results are compared with those reported by Suarez et al. for the hydrolytic catalyzed decompn. On the basis of these comparative studies, we propose a monodentate coordination of urea in the active site from which both the elimination and hydrolytic pathways can decomp. urea into CO2 and NH3. This observation is counter to what has been exptl. suggested based on the exogenous observation of carbamic acid (the reaction product from the hydrolysis pathway). However, this does not address what has occurred at the active site of urease prior to product release. On the basis of our computed results, the observation that urea prefers the elimination channel in aq. soln. and on the observation of Lippard and co-workers of an elimination reaction channel in a urease biomimetic model, we propose that the elimination channel needs to be re-examd. as a viable reaction channel in urease.
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    Carlsson, H.; Nordlander, E. Computational Modeling of the Mechanism of Urease. Bioinorg. Chem. Appl. 2010, 2010, 364891,  DOI: 10.1155/2010/364891
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    Barrios, A. M.; Lippard, S. J. Interaction of Urea with a Hydroxide-Bridged Dinuclear Nickel Center: An Alternative Model for the Mechanism of Urease. J. Am. Chem. Soc. 2000, 122, 9172– 9177,  DOI: 10.1021/ja000202v
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    Interaction of Urea with a Hydroxide-Bridged Dinuclear Nickel Center: An Alternative Model for the Mechanism of Urease
    Barrios, Amy M.; Lippard, Stephen J.
    Journal of the American Chemical Society (2000), 122 (38), 9172-9177CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    A hydroxide-bridged dinuclear nickel complex with a urea mol. linking the two metal ions through its carbonyl oxygen atom has been prepd. as a model for the metalloenzyme urease. This complex, [Ni2(μ-OH)(μ-urea)(bdptz)(urea)(CH3CN)](ClO4)3, where bdptz is the dinucleating ligand 1,4-bis(2,2'-dipyridylmethyl)phthalazine, effects the hydrolysis of urea upon heating in a two-step reaction. In the first step, a mol. of ammonia is eliminated from urea with concomitant prodn. of cyanate, the first-order rate const. in acetonitrile being (7.7 ± 0.5) × 10-4 h-1. This reaction is at least 500 times faster than the spontaneous decompn. of urea under the same conditions. When the cyanate-contg. product is further heated in the presence of water, the cyanate is hydrolyzed with a second-order rate const. of (9.5 ± 1) × 10-4 M-1 h-1. Reaction of [Ni2(μ-OH)(μ-urea)(bdptz)(urea)(CH3CN)](ClO4)3 in 50% aq. acetonitrile afforded ammonia with no appreciable buildup of the cyanate-contg. species. A possible analog of the cyanate-contg. product, [Ni2(μ-OH)(μ-H2O)(bdptz)(μ-OCN)]2(OTs)4, was independently synthesized and structurally characterized. These results establish the precedence for hydrolysis of urea via a cyanate intermediate as an alternative mechanism for the urease-catalyzed hydrolysis of urea.
  36. 36
    Senn, H. M.; Thiel, W. QM/MM Methods for Biomolecular Systems. Angew. Chem., Int. Ed. 2009, 48, 1198– 1229,  DOI: 10.1002/anie.200802019
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    QM/MM methods for biomolecular systems
    Senn, Hans Martin; Thiel, Walter
    Angewandte Chemie, International Edition (2009), 48 (7), 1198-1229CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Combined quantum-mechanics/mol.-mechanics (QM/MM) approaches have become the method of choice for modeling reactions in biomol. systems. Quantum-mech. (QM) methods are required for describing chem. reactions and other electronic processes, such as charge transfer or electronic excitation. However, QM methods are restricted to systems of up to a few hundred atoms. However, the size and conformational complexity of biopolymers calls for methods capable of treating up to several 100,000 atoms and allowing for simulations over time scales of tens of nanoseconds. This is achieved by highly efficient, force-field-based mol. mechanics (MM) methods. Thus to model large biomols. the logical approach is to combine the two techniques and, to use a QM method for the chem. active region (e.g., substrates and co-factors in an enzymic reaction) and an MM treatment for the surroundings (e.g., protein and solvent). The resulting schemes are commonly referred to as combined or hybrid QM/MM methods. They enable the modeling of reactive biomol. systems at a reasonable computational effort while providing the necessary accuracy.
  37. 37
    Ahmadi, S.; Barrios Herrera, L.; Chehelamirani, M.; Hostaš, J.; Jalife, S.; Salahub, D. R. Multiscale Modeling of Enzymes: QM-cluster, QM/MM, and QM/MM/MD: A Tutorial Review. Int. J. Quantum Chem. 2018, 118, e25558,  DOI: 10.1002/qua.25558
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    The ORCA program system
    Neese, Frank
    Wiley Interdisciplinary Reviews: Computational Molecular Science (2012), 2 (1), 73-78CODEN: WIRCAH; ISSN:1759-0884. (Wiley-Blackwell)
    A review. ORCA is a general-purpose quantum chem. program package that features virtually all modern electronic structure methods (d. functional theory, many-body perturbation and coupled cluster theories, and multireference and semiempirical methods). It is designed with the aim of generality, extendibility, efficiency, and user friendliness. Its main field of application is larger mols., transition metal complexes, and their spectroscopic properties. ORCA uses std. Gaussian basis functions and is fully parallelized. The article provides an overview of its current possibilities and documents its efficiency.
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    Becke, A. D. Density-functional Exchange-energy Approximation with Correct Asymptotic Behavior. Phys. Rev. A 1988, 38, 3098– 3100,  DOI: 10.1103/PhysRevA.38.3098
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    Density-functional exchange-energy approximation with correct asymptotic behavior
    Becke, A. D.
    Physical Review A: Atomic, Molecular, and Optical Physics (1988), 38 (6), 3098-100CODEN: PLRAAN; ISSN:0556-2791.
    Current gradient-cor. d.-functional approxns. for the exchange energies of at. and mol. systems fail to reproduce the correct 1/r asymptotic behavior of the exchange-energy d. A gradient-cor. exchange-energy functional is given with the proper asymptotic limit. This functional, contg. only one parameter, fits the exact Hartree-Fock exchange energies of a wide variety of at. systems with remarkable accuracy, surpassing the performance of previous functionals contg. two parameters or more.
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    Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-energy Formula into a Functional of the Electron Density. Phys. Rev. B 1988, 37, 785– 789,  DOI: 10.1103/PhysRevB.37.785
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    Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density
    Lee, Chengteh; Yang, Weitao; Parr, Robert G.
    Physical Review B: Condensed Matter and Materials Physics (1988), 37 (2), 785-9CODEN: PRBMDO; ISSN:0163-1829.
    A correlation-energy formula due to R. Colle and D. Salvetti (1975), in which the correlation energy d. is expressed in terms of the electron d. and a Laplacian of the 2nd-order Hartree-Fock d. matrix, is restated as a formula involving the d. and local kinetic-energy d. On insertion of gradient expansions for the local kinetic-energy d., d.-functional formulas for the correlation energy and correlation potential are then obtained. Through numerical calcns. on a no. of atoms, pos. ions, and mols., of both open- and closed-shell type, it is demonstrated that these formulas, like the original Colle-Salvetti formulas, give correlation energies within a few percent.
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    Becke, A. D. Density-functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. phys. 1993, 98, 5648– 5652,  DOI: 10.1063/1.464913
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    Density-functional thermochemistry. III. The role of exact exchange
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    Journal of Chemical Physics (1993), 98 (7), 5648-52CODEN: JCPSA6; ISSN:0021-9606.
    Despite the remarkable thermochem. accuracy of Kohn-Sham d.-functional theories with gradient corrections for exchange-correlation, the author believes that further improvements are unlikely unless exact-exchange information is considered. Arguments to support this view are presented, and a semiempirical exchange-correlation functional (contg. local-spin-d., gradient, and exact-exchange terms) is tested for 56 atomization energies, 42 ionization potentials, 8 proton affinities, and 10 total at. energies of first- and second-row systems. This functional performs better than previous functionals with gradient corrections only, and fits expt. atomization energies with an impressively small av. abs. deviation of 2.4 kcal/mol.
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    Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J. Ab Initio Calculation of Vibrational Absorption and Circular Dichroism Spectra Using Density Functional Force Fields. J. Phys. Chem. 1994, 98, 11623– 11627,  DOI: 10.1021/j100096a001
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    Ab Initio Calculation of Vibrational Absorption and Circular Dichroism Spectra Using Density Functional Force Fields
    Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J.
    Journal of Physical Chemistry (1994), 98 (45), 11623-7CODEN: JPCHAX; ISSN:0022-3654.
    The unpolarized absorption and CD spectra of the fundamental vibrational transitions of the chiral mol. 4-methyl-2-oxetanone are calcd. ab initio. Harmonic force fields are obtained using d. functional theory (DFT), MP2 and SCF methodologies, and a [5s4p2d/3s2p] (TZ2P) basis set. DFT calcns. use the LSDA, BLYP, and Becke3LYP (B3LYP) d. functionals. Mid-IR spectra predicted using LSDA, BLYP, and B3LYP force fields are of significantly different quality, the B3LYP force field yielding spectra in clearly superior, and overall excellent, agreement with expt. The MP2 force field yields spectra in slightly worse agreement with expt. than the B3LYP force field. The SCF force field yields spectra in poor agreement with expt. The basis set dependence of B3LYP force fields is also explored: the 6-31G* and TZ2P basis sets give very similar results while the 3-21G basis set yields spectra in substantially worse agreement with expt.
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    Neese, F.; Wennmohs, F.; Hansen, A.; Becker, U. Efficient, Approximate and Parallel Hartree-Fock and Hybrid DFT Calculations. A ‘Chain-of-Spheres’ Algorithm for the Hartree-Fock Exchange. Chem. Phys. 2009, 356, 98– 109,  DOI: 10.1016/j.chemphys.2008.10.036
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    Efficient, approximate and parallel Hartree-Fock and hybrid DFT calculations. A 'chain-of-spheres' algorithm for the Hartree-Fock exchange
    Neese, Frank; Wennmohs, Frank; Hansen, Andreas; Becker, Ute
    Chemical Physics (2009), 356 (1-3), 98-109CODEN: CMPHC2; ISSN:0301-0104. (Elsevier B.V.)
    In this paper, the possibility is explored to speed up Hartree-Fock and hybrid d. functional calcns. by forming the Coulomb and exchange parts of the Fock matrix by different approxns. For the Coulomb part the previously introduced Split-RI-J variant of the well-known d. fitting' approxn. is used. The exchange part is formed by semi-numerical integration techniques that are closely related to Friesner's pioneering pseudo-spectral approach. Our potentially linear scaling realization of this algorithm is called the 'chain-of-spheres exchange' (COSX). A combination of semi-numerical integration and d. fitting is also proposed. Both Split-RI-J and COSX scale very well with the highest angular momentum in the basis sets. It is shown that for extended basis sets speed-ups of up to two orders of magnitude compared to traditional implementations can be obtained in this way. Total energies are reproduced with an av. error of <0.3 kcal/mol as detd. from extended test calcns. with various basis sets on a set of 26 mols. with 20-200 atoms and up to 2000 basis functions. Reaction energies agree to within 0.2 kcal/mol (Hartree-Fock) or 0.05 kcal/mol (hybrid DFT) with the canonical values. The COSX algorithm parallelizes with a speedup of 8.6 obsd. for 10 processes. Min. energy geometries differ by less than 0.3 pm in the bond distances and 0.5° in the bond angles from their canonical values. These developments enable highly efficient and accurate SCF calcns. including nonlocal Hartree-Fock exchange for large mols. In combination with the RI-MP2 method and large basis sets, second-order many body perturbation energies can be obtained for medium sized mols. with unprecedented efficiency. The algorithms are implemented into the ORCA electronic structure system.

    Moving Frontiers in Quantum Chemistry.

  44. 44
    Weigend, F.; Ahlrichs, R. Balanced Basis Sets of Split Valence, Triple Zeta Valence and Quadruple Zeta Valence Quality for H to Rn: Design and Assessment of Accuracy. Phys. Chem. Chem. Phys. 2005, 7, 3297– 3305,  DOI: 10.1039/b508541a
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    Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy
    Weigend, Florian; Ahlrichs, Reinhart
    Physical Chemistry Chemical Physics (2005), 7 (18), 3297-3305CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
    Gaussian basis sets of quadruple zeta valence quality for Rb-Rn are presented, as well as bases of split valence and triple zeta valence quality for H-Rn. The latter were obtained by (partly) modifying bases developed previously. A large set of more than 300 mols. representing (nearly) all elements-except lanthanides-in their common oxidn. states was used to assess the quality of the bases all across the periodic table. Quantities investigated were atomization energies, dipole moments and structure parameters for Hartree-Fock, d. functional theory and correlated methods, for which we had chosen Moller-Plesset perturbation theory as an example. Finally recommendations are given which type of basis set is used best for a certain level of theory and a desired quality of results.
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    Breneman, C. M.; Wiberg, K. B. Determining Atom-centered Monopoles from Molecular Electrostatic Potentials. The Need for High Sampling Density in Formamide Conformational Analysis. J. Comput. Chem. 1990, 11, 361– 373,  DOI: 10.1002/jcc.540110311
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    Determining atom-centered monopoles from molecular electrostatic potentials. The need for high sampling density in formamide conformational analysis
    Breneman, Curt M.; Wiberg, Kenneth B.
    Journal of Computational Chemistry (1990), 11 (3), 361-73CODEN: JCCHDD; ISSN:0192-8651.
    An improved method for computing potential-derived charges is described which is based upon the CHELP program available from QCPE. This approach (CHELPG) is shown to be considerably less dependent upon mol. orientation than the original CHELP program. In the second part of this work, the CHELPG point selection algorithm was used to analyze the changes in the potential-derived charges in formamide during rotation about the C-N bond. In order to achieve a level of rotational invariance less than 10% of the magnitude of the electronic effects studied, an equally-spaced array of points 0.3 Å apart was required. Points found to be greater than 2.8 Å from any nucleus were eliminated, along with all points contained within the defined VDW distances from each of the atoms. The results are compared to those obtained by using CHELP. Even when large nos. of points (ca. 3000) were sampled using the CHELP selection routine, the results did not indicate a satisfactory level of rotational invariance. On the basis of these results, the original CHELP program was inadequate for analyzing internal rotations.
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    Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.; Mackerell, A. D., Jr. CHARMM General Force Field: A Force Field for Drug-like Molecules Compatible with the CHARMM All-atom Additive Biological Force Fields. J. Comput. Chem. 2010, 31, 671– 690,  DOI: 10.1002/jcc.21367
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    CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields
    Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.; Mackerell, A. D., Jr.
    Journal of Computational Chemistry (2010), 31 (4), 671-690CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
    The widely used CHARMM additive all-atom force field includes parameters for proteins, nucleic acids, lipids, and carbohydrates. In the present article, an extension of the CHARMM force field to drug-like mols. is presented. The resulting CHARMM General Force Field (CGenFF) covers a wide range of chem. groups present in biomols. and drug-like mols., including a large no. of heterocyclic scaffolds. The parametrization philosophy behind the force field focuses on quality at the expense of transferability, with the implementation concg. on an extensible force field. Statistics related to the quality of the parametrization with a focus on exptl. validation are presented. Addnl., the parametrization procedure, described fully in the present article in the context of the model systems, pyrrolidine, and 3-phenoxymethyl-pyrrolidine will allow users to readily extend the force field to chem. groups that are not explicitly covered in the force field as well as add functional groups to and link together mols. already available in the force field. CGenFF thus makes it possible to perform "all-CHARMM" simulations on drug-target interactions thereby extending the utility of CHARMM force fields to medicinally relevant systems. © 2009 Wiley Periodicals, Inc.J Comput Chem, 2010.
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    Li, P.; Roberts, B. P.; Chakravorty, D. K.; Merz, K. M., Jr. Rational Design of Particle Mesh Ewald Compatible Lennard-Jones Parameters for + 2 Metal Cations in Explicit Solvent. J. Chem. Theory Comput. 2013, 9, 2733– 2748,  DOI: 10.1021/ct400146w
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    Rational Design of Particle Mesh Ewald Compatible Lennard-Jones Parameters for +2 Metal Cations in Explicit Solvent
    Li, Pengfei; Roberts, Benjamin P.; Chakravorty, Dhruva K.; Merz, Kenneth M.
    Journal of Chemical Theory and Computation (2013), 9 (6), 2733-2748CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    Metal ions play significant roles in biol. systems. Accurate mol. dynamics (MD) simulations on these systems require a validated set of parameters. Although there are more detailed ways to model metal ions, the nonbonded model, which employs a 12-6 Lennard-Jones (LJ) term plus an electrostatic potential, is still widely used in MD simulations today due to its simple form. However, LJ parameters have limited transferability due to different combining rules, various water models, and diverse simulation methods. Recently, simulations employing a Particle Mesh Ewald (PME) treatment for long-range electrostatics have become more and more popular owing to their speed and accuracy. In the present work, we have systematically designed LJ parameters for 24 +2 metal (M(II)) cations to reproduce different exptl. properties appropriate for the Lorentz-Berthelot combining rules and PME simulations. We began by testing the transferability of currently available M(II) ion LJ parameters. The results showed that there are differences between simulations employing Ewald summation with other simulation methods and that it was necessary to design new parameters specific for PME based simulations. Employing the thermodn. integration (TI) method and performing periodic boundary MD simulations employing PME, allowed for a systematic investigation of the LJ parameter space. Hydration free energies (HFEs), the ion-oxygen distance in the first solvation shell (IOD), and coordination nos. (CNs) were obtained for various combinations of the parameters of the LJ potential for four widely used water models (TIP3P, SPC/E, TIP4P, and TIP4PEW). Results showed that the three simulated properties were highly correlated. Meanwhile, M(II) ions with the same parameters in different water models produce remarkably different HFEs but similar structural properties. It is difficult to reproduce various exptl. values simultaneously because the nonbonded model underestimates the interaction between the metal ions and water mols. at short-range. Moreover, the extent of underestimation increases successively for the TIP3P, SPC/E, TIP4PEW, and TIP4P water models. Nonetheless, we fitted a curve to describe the relationship between ε (the well depth) and radius (Rmin/2) from exptl. data on noble gases to facilitate the generation of the best possible compromise models. Hence, by targeting different exptl. values, we developed three sets of parameters for M(II) cations for three different water models (TIP3P, SPC/E, and TIP4PEW). These parameters we feel represent the best possible compromise that can be achieved using the nonbonded model for the ions in combination with simple water models. From a computational uncertainty anal. we est. that the uncertainty in our computed HFEs is on the order of ±1 kcal/mol. Further improvements will require more advanced nonbonded models likely with inclusion of polarization.
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    Lee, J.; Cheng, X.; Swails, J. M.; Yeom, M. S.; Eastman, P. K.; Lemkul, J. A.; Wei, S.; Buckner, J.; Jeong, J. C.; Qi, Y. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. J. Chem. Theory Comput. 2016, 12, 405– 413,  DOI: 10.1021/acs.jctc.5b00935
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    CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field
    Lee, Jumin; Cheng, Xi; Swails, Jason M.; Yeom, Min Sun; Eastman, Peter K.; Lemkul, Justin A.; Wei, Shuai; Buckner, Joshua; Jeong, Jong Cheol; Qi, Yifei; Jo, Sunhwan; Pande, Vijay S.; Case, David A.; Brooks, Charles L.; MacKerell, Alexander D.; Klauda, Jeffery B.; Im, Wonpil
    Journal of Chemical Theory and Computation (2016), 12 (1), 405-413CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    Proper treatment of nonbonded interactions is essential for the accuracy of mol. dynamics (MD) simulations, esp. in studies of lipid bilayers. The use of the CHARMM36 force field (C36 FF) in different MD simulation programs can result in disagreements with published simulations performed with CHARMM due to differences in the protocols used to treat the long-range and 1-4 nonbonded interactions. In this study, we systematically test the use of the C36 lipid FF in NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM. A wide range of Lennard-Jones (LJ) cutoff schemes and integrator algorithms were tested to find the optimal simulation protocol to best match bilayer properties of six lipids with varying acyl chain satn. and head groups. MD simulations of a 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) bilayer were used to obtain the optimal protocol for each program. MD simulations with all programs were found to reasonably match the DPPC bilayer properties (surface area per lipid, chain order parameters, and area compressibility modulus) obtained using the std. protocol used in CHARMM as well as from expts. The optimal simulation protocol was then applied to the other five lipid simulations and resulted in excellent agreement between results from most simulation programs as well as with exptl. data. AMBER compared least favorably with the expected membrane properties, which appears to be due to its use of the hard-truncation in the LJ potential vs. a force-based switching function used to smooth the LJ potential as it approaches the cutoff distance. The optimal simulation protocol for each program has been implemented in CHARMM-GUI. This protocol is expected to be applicable to the remainder of the additive C36 FF including the proteins, nucleic acids, carbohydrates, and small mols.
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    Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graph. 1996, 14, 33– 38,  DOI: 10.1016/0263-7855(96)00018-5
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    VDM: visual molecular dynamics
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    Journal of Molecular Graphics (1996), 14 (1), 33-8, plates, 27-28CODEN: JMGRDV; ISSN:0263-7855. (Elsevier)
    VMD is a mol. graphics program designed for the display and anal. of mol. assemblies, in particular, biopolymers such as proteins and nucleic acids. VMD can simultaneously display any no. of structures using a wide variety of rendering styles and coloring methods. Mols. are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resoln. raster images of displayed mols. may be produced by generating input scripts for use by a no. of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate mol. dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biol., which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs, VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
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    Park, I.-S.; Hausinger, R. P. Site-Directed Mutagenesis of Klebsiella aerogenes Urease: Identification of Histidine Residues that Appear to Function in Nickel Ligation, Substrate Binding, and Catalysis. Protein Sci. 1993, 2, 1034– 1041,  DOI: 10.1002/pro.5560020616
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    Site-directed mutagenesis of Klebsiella aerogenes urease: Identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis
    Park, Il Seon; Hausinger, Robert P.
    Protein Science (1993), 2 (6), 1034-41CODEN: PRCIEI; ISSN:0961-8368.
    Comparison of six urease sequences revealed the presence of 10 conserved histidine residues (H96 in the γ subunit, H39 and H41 in β, and H134, H136, H219, H246, H312, H320, and H321 in the α subunit of the K. aerogenes enzyme). Each of these residues in K. aerogenes urease was substituted with alanine by site-directed mutagenesis, and the mutant proteins were purified and characterized in order to identify essential histidine residues and assign their roles. The γH96A, βH39A, βH41A, αH312A, and αH321A mutant proteins possess activities and nickel contents similar to wild-type enzyme, suggesting that these residues are not essential for substrate binding, catalysis, or metal binding. In contrast, the αH134A, αH136A, and αH246A proteins exhibit no detectable activity and possess 53%, 6%, and 21% of the nickel content of wild-type enzyme. These results are consistent with αH134, αH136, and αH246 functioning as nickel ligands. The αH219A protein is active and has nickel (∼1.9% and ∼80%, resp., when compared to wild-type protein) but exhibits a very high Km value (1,100 mM compared to 2.3 mM for the wild-type enzyme). These results are compatible with αH219 having some role in facilitating substrate binding. Finally, the αH320A protein (Km = 8.3 mM) only displays ∼0.003% of the wild-type enzyme activity, despite having a normal nickel content. Unlike the wild-type and αH219A ureases, this mutant protein was not inactivated by diethylpyrocarbonate (DEP), consistent with αH320 being the DEP-reactive general base that facilitates catalysis.
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    Nelson, M. T.; Humphrey, W.; Gursoy, A.; Dalke, A.; Kalé, L. V.; Skeel, R. D.; Schulten, K. NAMD: A Parallel, Object-Oriented Molecular Dynamics Program. Int. J. High Perform. Comput. Appl. 1996, 10, 251– 268,  DOI: 10.1177/109434209601000401
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    Darden, Tom; York, Darrin; Pedersen, Lee
    Journal of Chemical Physics (1993), 98 (12), 10089-92CODEN: JCPSA6; ISSN:0021-9606.
    An N·log(N) method for evaluating electrostatic energies and forces of large periodic systems is presented. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolution using fast Fourier transforms. Timings and accuracies are presented for three large cryst. ionic systems.
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    Huang, J.; MacKerell, A. D., Jr CHARMM36 All-atom Additive Protein Force Field: Validation Based on Comparison to NMR Data. J. Comput. Chem. 2013, 34, 2135– 2145,  DOI: 10.1002/jcc.23354
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    CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data
    Huang, Jing; MacKerell, Alexander D. Jr
    Journal of Computational Chemistry (2013), 34 (25), 2135-2145CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
    Protein structure and dynamics can be characterized on the atomistic level with both NMR expts. and mol. dynamics (MD) simulations. Here, the authors quantify the ability of the recently presented CHARMM36 (C36) force field (FF) to reproduce various NMR observables using MD simulations. The studied NMR properties include backbone scalar couplings across hydrogen bonds, residual dipolar couplings (RDCs) and relaxation order parameter, as well as scalar couplings, RDCs, and order parameters for side-chain amino- and methyl-contg. groups. The C36 FF leads to better correlation with exptl. data compared to the CHARMM22/CMAP FF and suggest using C36 in protein simulations. Although both CHARMM FFs contains the same nonbond parameters, the authors' results show how the changes in the internal parameters assocd. with the peptide backbone via CMAP and the χ1 and χ2 dihedral parameters leads to improved treatment of the analyzed nonbond interactions. This highlights the importance of proper treatment of the internal covalent components in modeling nonbond interactions with mol. mechanics FFs.
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    Bannwarth, C.; Ehlert, S.; Grimme, S. GFN2-xTB─An Accurate and Broadly Parametrized Self-Consistent Tight-Binding Quantum Chemical Method with Multipole Electrostatics and Density-Dependent Dispersion Contributions. J. Chem. Theory Comput. 2019, 15, 1652– 1671,  DOI: 10.1021/acs.jctc.8b01176
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    GFN2-xTB-An Accurate and Broadly Parametrized Self-Consistent Tight-Binding Quantum Chemical Method with Multipole Electrostatics and Density-Dependent Dispersion Contributions
    Bannwarth, Christoph; Ehlert, Sebastian; Grimme, Stefan
    Journal of Chemical Theory and Computation (2019), 15 (3), 1652-1671CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
    An extended semiempirical tight-binding model is presented, which is primarily designed for the fast calcn. of structures and noncovalent interactions energies for mol. systems with roughly 1000 atoms. The essential novelty in this so-called GFN2-xTB method is the inclusion of anisotropic second order d. fluctuation effects via short-range damped interactions of cumulative at. multipole moments. Without noticeable increase in the computational demands, this results in a less empirical and overall more phys. sound method, which does not require any classical halogen or hydrogen bonding corrections and which relies solely on global and element-specific parameters (available up to radon, Z = 86). Moreover, the at. partial charge dependent D4 London dispersion model is incorporated self-consistently, which can be naturally obtained in a tight-binding picture from second order d. fluctuations. Fully anal. and numerically precise gradients (nuclear forces) are implemented. The accuracy of the method is benchmarked for a wide variety of systems and compared with other semiempirical methods. Along with excellent performance for the "target" properties, we also find lower errors for "off-target" properties such as barrier heights and mol. dipole moments. High computational efficiency along with the improved physics compared to it precursor GFN-xTB makes this method well-suited to explore the conformational space of mol. systems. Significant improvements are furthermore obsd. for various benchmark sets, which are prototypical for biomol. systems in aq. soln.
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    Melo, M. C.; Bernardi, R. C.; Rudack, T.; Scheurer, M.; Riplinger, C.; Phillips, J. C.; Maia, J. D.; Rocha, G. B.; Ribeiro, J. V.; Stone, J. E. NAMD Goes Quantum: An Integrative Suite for Hybrid Simulations. Nat. Methods 2018, 15, 351– 354,  DOI: 10.1038/nmeth.4638
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    Maruno, Y.; Shoji, M.; Koizumi, K.; Nishiyama, Y.; Kitagawa, Y.; Kawakami, T.; Okumura, M.; Yamaguchi, K. Theoretical Studies on Magnetic Interactions between Ni (II) Ions in Urease. Polyhedron 2005, 24, 2778– 2783,  DOI: 10.1016/j.poly.2005.03.161
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    Theoretical studies on magnetic interactions between Ni(II) ions in urease
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    Polyhedron (2005), 24 (16-17), 2778-2783CODEN: PLYHDE; ISSN:0277-5387. (Elsevier B.V.)
    The chem. species of bridge oxygen (WB) in active site of urease was examd. in terms of a magnetic interaction between Ni(II) ions. The effective exchange integrals (Jab) values between Ni(II) ions were calcd. by using three candidates of WB, i.e., H2O, OH- and O2-. The unrestricted hybrid d. functional theory (UHDFT) calcns. were carried out using the smallest models and realistic models. In the case of the smallest models, the exptl. Jab value (-6.3 cm-1) was a middle value of WB = H2O (-2.6 cm-1) and WB = OH- (-11.4 cm-1), while we could not obtain an appropriate electronic structure using the O2--bridge model. The Jab became -16.7 cm-1 by using more realistic OH--bridge model, while one spin d. on the Ni2 ion was delocalized to Asp360 in case of H2O-bridge model. Natural orbital analyses revealed that the magnetic orbital through Lys-bridge delocalized to Asp360 and it seemed to assoc. with the spin transfer. From the results, the OH--bridge seemed to be the most likely candidate for the structure of active site in urease.
  58. 58
    Kitagawa, Y.; Saito, T.; Ito, M.; Nakanishi, Y.; Shoji, M.; Koizumi, K.; Yamanaka, S.; Kawakami, T.; Okumura, M.; Yamaguchi, K. Geometry Optimization Method Based on Approximate Spin Projection and its Application to F2, CH2, CH2OO, and Active Site of Urease. Int. J. Quantum Chem. 2007, 107, 3094– 3102,  DOI: 10.1002/qua.21456
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    Geometry optimization method based on approximate spin projection and its application to F2, CH2, CH2OO, and active site of urease
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    A new geometry optimization method based on an approx. spin projection (AP) procedure is proposed to eliminate a spin contamination effect in an optimized structure on a low spin (LS) state of a broken symmetry (BS) method. First, an energy gradient with the AP (AP gradient) is derived and it is applied to the geometry optimization of F2, CH2, and CH2OO in order to obtain their structures without the spin contamination. The optimization method corrects H-C-H angle of the CH2 about 10° in comparison with the BS method, and indicates that the spin contamination in optimized geometry of the BS LS states is considerably large. Next, an active site of Urease that consists of two Ni(II) ions is optimized by the AP method. The result indicates that structure of the active site is sensitive to a water mol. called W3.
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    Temperature- and pressure-dependent stopped-flow kinetic studies of jack bean urease. Implications for the catalytic mechanism
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    Urease, a Ni-contg. metalloenzyme, features an activity that has profound medical and agricultural implications. The mechanism of this activity, however, has not been as yet thoroughly established. Accordingly, to improve its understanding, in this study we analyzed the steady-state kinetic parameters of the enzyme (jack bean), Km and kcat, measured at different temps. and pressures. Such an anal. is useful as it provides information on the mol. nature of the intermediate and transition states of the catalytic reaction. We measured the parameters in a noninteracting buffer using a stopped-flow technique at 15-35° and in the pressure range 5-132 MPa, the pressure-dependent measurements being the first of their kind performed for urease. While temp. enhanced the activity of urease, pressure inhibited the enzyme; the inhibition was biphasic. Analyzing Km provided the characteristics of the formation of the ES complex, and analyzing kcat, the characteristics of the activation of ES. From the temp.-dependent measurements, the energetic parameters were derived, i.e. thermodn. ΔHo and ΔSo for ES formation, and kinetic ΔH≠ and ΔS≠ for ES activation, while from the pressure-dependent measurements, the binding ΔVb and activation vols. were detd. The thermodn. and activation parameters obtained are discussed in terms of the current proposals for the mechanism of the urease reaction, and they are found to support the mechanism proposed by Benini et al., in which the Ni-Ni bridging hydroxide-not the terminal hydroxide-is the nucleophile in the catalytic reaction.
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    El-Hefnawy, M. E.; Sakran, M.; Ismail, A. I.; Aboelfetoh, E. F. Extraction, Purification, Kinetic and Thermodynamic Properties of Urease from Germinating Pisum Sativum L. seeds. BMC Biochem. 2014, 15, 15,  DOI: 10.1186/1471-2091-15-15
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    Extraction, purification, kinetic and thermodynamic properties of urease from germinating Pisum sativum L. seeds
    El-Hefnawy, Mohamed E.; Sakran, Mohamed; Ismail, Ali I.; Aboelfetoh, Eman Fahmy
    BMC Biochemistry (2014), 15 (), 15/1-15/8, 8 pp.CODEN: BBMIB3 ISSN:. (BioMed Central Ltd.)
    Background: Urease, one of the highly efficient known enzymes, catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The present study aimed to ext. urease from pea seeds (Pisum sativum L). The enzyme was then purified in three consequence steps: acetone pptn., DEAE-cellulose ion-exchange chromatog. and gel filtration chromatog. (Sephacryl S-200 column). Results: The purifn. fold was 12.85 with a yield of 40%. The mol. wt. of the isolated urease was estd. by chromatog. to be 269,000 Daltons. Maximum urease activity (190 U/g) was achieved at the optimum conditions of 40°C and pH of 7.5 after 5 min of incubation. The kinetic parameters, Km and Vmax, were estd. by Lineweaver-Burk fits and found to be 500 mM and 333.3 U/g, resp. The thermodn. consts. of activation, ΔH, Ea, and ΔS, were detd. using Arrhenius plot and found to be 21.20 kJ/mol, 23.7 kJ/mol and 1.18 kJ/mol/K, resp. Conclusions: Urease was purified from germinating Pisum sativum L. seeds. The purifn. fold, yield and mol. wt. were detd. The effects of pH, concn. of enzyme, temp., concn. of substrate and storage period on urease activity were examd. This may provide an insight on the various aspects of the property of the enzyme. The significance of extg. urease from different sources could play a good role in understanding the metab. of urea in plants.
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    Musiani, F.; Arnofi, E.; Casadio, R.; Ciurli, S. Structure-Based Computational Study of the Catalytic and Inhibition Mechanisms of Urease. J. Biol. Inorg. Chem. 2001, 6, 300– 314,  DOI: 10.1007/s007750000204
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    Structure-based computational study of the catalytic and inhibition mechanisms of urease
    Musiani, Francesco; Arnofi, Elisa; Casadio, Rita; Ciurli, Stefano
    JBIC, Journal of Biological Inorganic Chemistry (2001), 6 (3), 300-314CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)
    The viability of different mechanisms of catalysis and inhibition of the nickel-contg. enzyme urease was explored using the available high-resoln. structures of the enzyme isolated from Bacillus pasteurii in the native form and inhibited with several substrates. The structures and charge distribution of urea, its catalytic transition state, and three enzyme inhibitors were calcd. using ab initio and d. functional theory methods. The DOCK program suite was employed to det. families of structures of urease complexes characterized by docking energy scores indicative of their relative stability according to steric and electrostatic criteria. Adjustment of the parameters used by DOCK, in order to account for the presence of the metal ion in the active site, resulted in the calcn. of best energy structures for the nickel-bound inhibitors β-mercaptoethanol, acetohydroxamic acid, and diamidophosphoric acid. These calcd. structures are in good agreement with the exptl. detd. structures, and provide hints on the reactivity and mobility of the inhibitors in the active site. The same docking protocol was applied to the substrate urea and its catalytic transition state, in order to shed light onto the possible catalytic steps occurring at the binuclear nickel active site. These calcns. suggest that the most viable pathway for urea hydrolysis involve a nucleophilic attack by the bridging, and not the terminal, nickel-bound hydroxide onto a urea mol., with active site residues playing important roles in orienting and activating the substrate, and stabilizing the catalytic transition state.

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  • Abstract

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    Scheme 1

    Scheme 1. Urea Hydrolysis Reaction
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    Figure 1

    Figure 1. Active site of Sporosarcina pasteurii urease (SPU) and two possible substrate binding modes, monodentate 1 and bidentate 2.

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    Scheme 2

    Scheme 2. Previously Proposed Urease Reaction Mechanisms (i)–(iv) and Our Proposed One (v), the Details of Which Are Described in the Text, with Amino Acid Numbering for SPU/KAU
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    Figure 2

    Figure 2. QM regions of 1 and 2 after the QM(GFN2-xTB)/MM MD equilibrations (top). Key bond distances are also presented in Å (bottom).

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    Figure 3

    Figure 3. Potential energy profiles (in kcal mol–1) and illustrations of the transition state and product structures corresponding to (a) mechanism (v) starting from 1′ and (b) mechanism (ii) starting from 2′ obtained at GFN2-xTB and UB3LYP/def2-SVP levels.

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    Figure 4

    Figure 4. (a) One-dimensional potentials of mean force (1D-PMF) of the nucleophilic attack reaction and representative snapshots of the active site in (b) 1R, (c) 1TS1A, and (d) 1IntA, with key distances in Å.

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    Figure 5

    Figure 5. (a) One-dimensional potentials of mean force (1D-PMF) of the proton transfer reaction and representative snapshots of the active site in (b) 1TS2A and (c) 1PA, with key distances in Å.

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    Figure 6

    Figure 6. (a) One-dimensional potentials of mean force (1D-PMF) of the W2 dissociation and representative snapshots of the active site in (b) 1TSB and (c) 1PB, with key distances in Å.

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    Figure 7

    Figure 7. (a) One-dimensional potentials of mean force (1D-PMF) of the nucleophilic attack reaction and representative snapshots of the active site in (b) 2R, (c) 2TS1A, and (d) 2IntA, with key distances in Å.

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    Figure 8

    Figure 8. (a) One-dimensional potentials of mean force (1D-PMF) of the proton transfer reaction and representative snapshots of the active site in (b) 2TS2A and (c) 2PA, with key distances in Å.

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  • References

    This article references 64 other publications.

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      Blakeley, R. L.; Webb, E. C.; Zerner, B. Jack Bean Urease (EC 3.5.1.5). A New Purification and Reliable Rate Assay. Biochemistry 1969, 8, 1984– 1990,  DOI: 10.1021/bi00833a031
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      Jack bean urease (EC 3.5.1.5) was reproducibly purified to a hitherto unobtained and const. specific activity. The procedure is capable of handling "poor" jack bean meal. A CHCl3-Me2CO powder of jack bean meal is extd. with 30% Me2CO contg. 1% mercaptoethanol at 39° for 5 min. The filtrate from this step is allowed to stand for 48 hrs. at 4°, and the crystals of the enzyme are harvested by centrifugation. After recycling gel filtration in the presence of 1 mM mercaptoethanol and 1 mM EDTA (Sephadex G-200), and concn. by (NH4)2SO4 pptn., the enzyme reached max. specific activity. A reliable pH-stat assay in the presence of 2 μM dithiothreitol was devised.
    6. 6
      Carter, E. L.; Flugga, N.; Boer, J. L.; Mulrooney, S. B.; Hausinger, R. P. Interplay of Metal Ions and Urease. Metallomics 2009, 1, 207– 221,  DOI: 10.1039/b903311d
      6
      Interplay of metal ions and urease
      Carter, Eric L.; Flugga, Nicholas; Boer, Jodi L.; Mulrooney, Scott B.; Hausinger, Robert P.
      Metallomics (2009), 1 (3), 207-221CODEN: METAJS; ISSN:1756-591X. (Royal Society of Chemistry)
      A review. Urease, the first enzyme to be crystd., contains a dinuclear nickel metallocenter that catalyzes the decompn. of urea to produce ammonia, a reaction of great agricultural and medical importance. Several mechanisms of urease catalysis have been proposed on the basis of enzyme crystal structures, model complexes, and computational efforts, but the precise steps in catalysis and the requirement of nickel vs. other metals remain unclear. Purified bacterial urease is partially activated via incubation with carbon dioxide plus nickel ions; however, in vitro activation also has been achieved with manganese and cobalt. In vivo activation of most ureases requires accessory proteins that function as nickel metallochaperones and GTP-dependent mol. chaperones or play other roles in the maturation process. In addn., some microorganisms control their levels of urease by metal ion-dependent regulatory mechanisms.
    7. 7
      Zambelli, B.; Musiani, F.; Benini, S.; Ciurli, S. Chemistry of Ni2+ in Urease: Sensing, Trafficking, and Catalysis. Acc. Chem. Res. 2011, 44, 520– 530,  DOI: 10.1021/ar200041k
      7
      Chemistry of Ni2+ in urease: Sensing, trafficking, and catalysis
      Zambelli, Barbara; Musiani, Francesco; Benini, Stefano; Ciurli, Stefano
      Accounts of Chemical Research (2011), 44 (7), 520-530CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Transition metals are both essential to enzymic catalysis and limited in environmental availability. These 2 biol. facts have together driven organisms to evolve mechanisms for selective metal ion sensing and utilization. Changes in metal ion concns. are perceived by metal-dependent transcription factors and transduced into appropriate cellular responses, which regulate the machineries of competitive metal ion homeostasis and metalloenzyme activation. The intrinsic toxicity of the majority of metal ions further creates a need for regulated intracellular trafficking, which is carried out by specific chaperones. The Ni2+-dependent urease system serves as a paradigm for studying the strategies that cells use to handle an essential, yet toxic, metal ion. Although the discovery of urease as the 1st biol. system for which Ni2+ is essential for activity dates to 1975, the rationale for Ni2+ selection, as well as the cascade of events involving metal-dependent gene regulation and protein-protein interactions leading to enzyme activation, have yet to be fully unraveled. The past 14 years since the paper by P. A. Karplus et al. (1997) have witnessed impressive achievements in the understanding of the biochem. of Ni2+ in the urease system. Here, the authors discuss more recent advances in the comprehension of the specific role of Ni2+ in catalysis and in the interplay between Ni2+ and other metal ions, such as Zn2+ and Fe2+, in metal-dependent enzyme activity. The discussion focuses on work carried out in the authors' lab. In particular, the structural features of the enzyme bound to inhibitors, substrate analogs, and transition state or intermediate analogs have shed light on the catalytic mechanism. Structural and functional information has been correlated to understand the Ni2+ sensing effected by NikR protein, a Ni2+-dependent transcription factor. The urease activation process, involving the insertion of Ni2+ into the urease active site, has been in part dissected and analyzed through the investigation of the mol. properties of accessory proteins UreD, UreF, and UreG. The intracellular trafficking of Ni2+ has been rationalized through a deeper understanding of the structural and metal-binding properties of metallochaperone UreE. All the while, a no. of key general concepts have been revealed and developed. These include an understanding of the following: (1) the overall ancillary role of Zn2+ in Ni metab.; (2) the intrinsically disordered nature of the GTPase responsible for coupling the energy consumption to the CO2 requirement for the urease activation process; and (3) the role of the accessory proteins regulating this GTPase activity.
    8. 8
      Maroney, M. J.; Ciurli, S. Nonredox Nickel Enzymes. Chem. Rev. 2014, 114, 4206– 4228,  DOI: 10.1021/cr4004488
      8
      Nonredox nickel enzymes
      Maroney, Michael J.; Ciurli, Stefano
      Chemical Reviews (Washington, DC, United States) (2014), 114 (8), 4206-4228CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Current knowledge of the biochem., structure, and catalytic mechanism of enzymes, including urease, glyoxalase I, and acireductone dioxygenase, whose active sites require Ni and utilize it in a non-redox role, are discussed.
    9. 9
      Mazzei, L.; Musiani, F.; Ciurli, S. The Structure-based Reaction Mechanism of Urease, a Nickel Dependent Enzyme: Tale of a Long Debate. J. Biol. Inorg. Chem. 2020, 25, 829,  DOI: 10.1007/s00775-020-01808-w
      9
      The structure-based reaction mechanism of urease, a nickel dependent enzyme: tale of a long debate
      Mazzei, Luca; Musiani, Francesco; Ciurli, Stefano
      JBIC, Journal of Biological Inorganic Chemistry (2020), 25 (6), 829-845CODEN: JJBCFA; ISSN:0949-8257. (Springer)
      Abstr.: This review is an attempt to retrace the chronicle that starts from the discovery of the role of nickel as the essential metal ion in urease for the enzymic catalysis of urea, a key step in the biogeochem. cycle of nitrogen on Earth, to the most recent progress in understanding the chem. of this historical enzyme. Data and facts are presented through the magnifying lenses of the authors, using their best judgment to filter and elaborate on the many facets of the research carried out on this metalloenzyme over the years. The tale is divided in chapters that discuss and describe the results obtained in the subsequent leaps in the knowledge that led from the discovery of a biol. role for Ni to the most recent advancements in the comprehension of the relationship between the structure and function of urease. This review is intended not only to focus on the bioinorg. chem. of this beautiful metal-based catalysis, but also, and maybe primarily, to evoke inspiration and motivation to further explore the realm of bio-based coordination chem.
    10. 10
      Konieczna, I.; Zarnowiec, P.; Kwinkowski, M.; Kolesinska, B.; Fraczyk, J.; Kaminski, Z.; Kaca, W. Bacterial urease and its role in long-lasting human diseases. Curr. Protein Pept. Sci. 2012, 13, 789– 806,  DOI: 10.2174/138920312804871094
      10
      Bacterial urease and its role in long-lasting human diseases
      Konieczna, Iwona; Zarnowiec, Paulina; Kwinkowski, Marek; Kolesinska, Beata; Fraczyk, Justyna; Kaminski, Zbigniew; Kaca, Wieslaw
      Current Protein and Peptide Science (2012), 13 (8), 789-806CODEN: CPPSCM; ISSN:1389-2037. (Bentham Science Publishers Ltd.)
      A review. Urease is a virulence factor found in various pathogenic bacteria. It is essential in colonization of a host organism and in maintenance of bacterial cells in tissues. Due to its enzymic activity, urease has a toxic effect on human cells. The presence of ureolytic activity is an important marker of a no. of bacterial infections. Urease is also an immunogenic protein and is recognized by antibodies present in human sera. The presence of such antibodies is connected with progress of several long-lasting diseases, like rheumatoid arthritis, atherosclerosis or urinary tract infections. In bacterial ureases, motives with a sequence and/or structure similar to human proteins may occur. This phenomenon, known as mol. mimicry, leads to the appearance of autoantibodies, which take part in host mols. destruction. Detection of antibodies-binding motives (epitopes) in bacterial proteins is a complex process. However, org. chem. tools, such as synthetic peptide libraries, are helpful in both, epitope mapping as well as in serol. investigations. In this review, we present a synthetic report on a mol. organization of bacterial ureases - genetic as well as structural. We characterize methods used in detecting urease and ureolytic activity, including techniques applied in disease diagnostic processes and in chem. synthesis of urease epitopes. The review also provides a summary of knowledge about a toxic effect of bacterial ureases on human body and about occurrence of anti-urease antibodies in long-lasting diseases.
    11. 11
      Rajakovich, L. J.; Balskus, E. P. Metabolic Functions of the Human Gut Microbiota: the Role of Metalloenzymes. Nat. Prod. Rep. 2019, 36, 593– 625,  DOI: 10.1039/C8NP00074C
      11
      Metabolic functions of the human gut microbiota: the role of metalloenzymes
      Rajakovich, Lauren J.; Balskus, Emily P.
      Natural Product Reports (2019), 36 (4), 593-625CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      Covering: up to the end of 2017The human body is composed of an equal no. of human and microbial cells. While the microbial community inhabiting the human gastrointestinal tract plays an essential role in host health, these organisms have also been connected to various diseases. Yet, the gut microbial functions that modulate host biol. are not well established. In this review, we describe metabolic functions of the human gut microbiota that involve metalloenzymes. These activities enable gut microbial colonization, mediate interactions with the host, and impact human health and disease. We highlight cases in which enzyme characterization has advanced our understanding of the gut microbiota and examples that illustrate the diverse ways in which metalloenzymes facilitate both essential and unique functions of this community. Finally, we analyze Human Microbiome Project sequencing datasets to assess the distribution of a prominent family of metalloenzymes in human-assocd. microbial communities, guiding future enzyme characterization efforts.
    12. 12
      Bremner, J. M.; Krogmeier, M. J. Evidence that the Adverse Effect of Urea Fertilizer on Seed Germination in Soil is due to Ammonia Formed through Hydrolysis of Urea by Soil Urease. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 8185– 8188,  DOI: 10.1073/pnas.86.21.8185
      12
      Evidence that the adverse effect of urea fertilizer on seed germination in soil is due to ammonia formed through hydrolysis of urea by soil urease
      Bremner, John M.; Krogmeier, Michael J.
      Proceedings of the National Academy of Sciences of the United States of America (1989), 86 (21), 8185-8CODEN: PNASA6; ISSN:0027-8424.
      Studies using seeds of wheat (Triticum aestivum), rye (Secale cereale), barley (Hordeum vulgare) and corn (Zea mays) indicated that the adverse effect of urea fertilizer on seed germination in soil is due to ammonia formed through hydrolysis of urea by soil urease and is not due to urea itself, to urea fertilizer impurities such as biuret, or to nitrite formed by nitrification of urea nitrogen. Support for this conclusion was obtained from (i) comparison of the effects on seed germination in soil of purified urea, urea fertilizers, urea fertilizer impurities, and compds. formed by enzymic and microbial transformations of urea in soil; (ii) studies showing that ammonia volatilized from soils treated with urea completely inhibited germination of seeds close to, but not in contact with, these soils; and (iii) expts. showing that the adverse effect of urea fertilizer on seed germination in soil was completely eliminated when the soil was autoclaved to destroy urease or was treated with phenylphosphorodiamidate to inhibit soil urease activity before treatment with urea fertilizer.
    13. 13
      Gioacchini, P.; Nastri, A.; Marzadori, C.; Giovannini, C.; Antisari, L. V.; Gessa, C. Influence of Urease and Nitrification Inhibitors on N Losses from Soils Fertilized with Urea. Biol. Fertil. 2002, 36, 129– 135,  DOI: 10.1007/s00374-002-0521-1
      13
      Influence of urease and nitrification inhibitors on N losses from soils fertilized with urea
      Gioacchini, Paola; Nastri, Anna; Marzadori, Claudio; Giovannini, Camilla; Vittori Antisari, Livia; Gessa, Carlo
      Biology and Fertility of Soils (2002), 36 (2), 129-135CODEN: BFSOEE; ISSN:0178-2762. (Springer-Verlag)
      The aim was to evaluate how the N losses through volatilization and leaching from soils fertilized with urea can be affected by the application of a urease inhibitor or a urease plus a nitrification inhibitor. The expt. was carried out using lysimeters with 15N-labeled urea and N-butylthiophosphoric triamide (NBPT) as urease inhibitor and dicyandiamide (DCD) as nitrification inhibitor, comparing 3 treatments: urea alone (U), urea + NBPT (UN) and urea + NBPT + DCD (UND). Both volatilization and leaching were significantly different in the soils used, according to their physicochem. characteristics. However, the pattern of the loss was similar: the volatilization was significantly reduced by NBPT (UN), but the presence of DCD (UND) significantly increased the loss, with respect to UN. Considering leaching, the highest amt. of NO3- was lost with UND, the lowest with U. The greatest amt. of N lost by leaching was soil-derived N produced by the N mineralization-immobilization turnover. We suggest that, by maintaining the NH4+ in the soils, the inhibitors, in particular DCD, caused a priming effect with a subsequent increase in the rate of soil org. matter mineralization and an extra release of soil org. N. The priming effect was real in the sandy loam (SL) soil where a net N release was obsd., whereas in the clay loam (CL) soil the effect of the inhibitors was less pronounced and an apparent priming effect was obsd.; however, a real priming effect also cannot be excluded in this soil.
    14. 14
      Jabri, E.; Carr, M. B.; Hausinger, R. P.; Karplus, P. A. The Crystal Structure of Urease from Klebsiella aerogenes. Science 1995, 268, 998– 1004,  DOI: 10.1126/science.7754395
      14
      The crystal structure of urease from Klebsiella aerogenes
      Jabri, Evelyn; Carr, Mary Beth; Hausinger, Robert P.; Karplus, P. Andrew
      Science (Washington, D. C.) (1995), 268 (5213), 998-1004CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The crystal structure of urease from K. aerogenes was detd. at 2.2 Å resoln. and refined to an R factor of 18.2%. The enzyme contained 4 structural domains: 3 with novel folds playing structural roles, and an (αβ)8 barrel domain, which contained the bi-Ni center. The 2 active site Ni ions were 3.5 Å apart. One Ni ion was coordinated by 3 ligands (with low occupancy of a 4th ligand) and the 2nd was coordinated by 5 ligands. A carbamylated Lys residue provided an O ligand to each Ni, explaining why CO2 is required for the activation of urease apoenzyme. The structure was compatible with a catalytic mechanism whereby urea ligates Ni-1 to complete its tetrahedral coordination and a hydroxide ligand of Ni-2 attacks the carbonyl C atom. A surprisingly high structural similarity between the urease catalytic domain and that of the Zn-dependent adenosine deaminase revealed a remarkable example of active site divergence.
    15. 15
      Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Miletti, S.; Ciurli, S.; Mangani, S. A New Proposal for Urease Mechanism Based on the Crystal Structures of the Native and Inhibited Enzyme from Bacillus pasteurii: Why Urea Hydrolysis Costs Two Nickels. Structure 1999, 7, 205– 216,  DOI: 10.1016/S0969-2126(99)80026-4
      15
      A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels
      Benini, Stefano; Rypniewski, Wojciech R.; Wilson, Keith S.; Miletti, Silvia; Ciurli, Stefano; Mangani, Stefano
      Structure (London) (1999), 7 (2), 205-216CODEN: STRUE6; ISSN:0969-2126. (Current Biology Publications)
      Urease catalyzes the hydrolysis of urea, the final step of org. nitrogen mineralization, using a bimetallic nickel center. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are assocd. with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a redn. of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the mol. level. The structures of native and inhibited urease from Bacillus pasteurii have been detd. at a resoln. of 2.0 Å by synchroton X-ray cryogenic crystallog. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water mol. and a bridging hydroxide. A fourth water mol. completes a tetrahedral cluster of solvent mols. The enzyme crystd. in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analog diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymic urea hydrolysis which reconciles the available structural and biochem. data.
    16. 16
      Ha, N.-C.; Oh, S.-T.; Sung, J. Y.; Cha, K. A.; Lee, M. H.; Oh, B.-H. Supramolecular Assembly and Acid Resistance of Helicobacter pylori Urease. Nat. Struct. Biol. 2001, 8, 505– 509,  DOI: 10.1038/88563
      16
      Supramolecular assembly and acid resistance of Helicobacter pylori urease
      Ha, Nam-Chul; Oh, Sang-Taek; Sung, Jae Young; Cha, Kyeung Ah; Lee, Mann Hyung; Oh, Byung-Ha
      Nature Structural Biology (2001), 8 (6), 505-509CODEN: NSBIEW; ISSN:1072-8368. (Nature America Inc.)
      H. pylori, an etiol. agent in a variety of gastro-duodenal diseases, produces a large amt. of urease (I), which is believed to neutralize gastric acid by producing NH3 for the survival of the bacteria. Up to 30% of I assocs. with the surface of intact cells upon lysis of neighboring bacteria. The role of I at the extracellular location has been a subject of controversy because the purified enzyme is irreversibly inactivated below pH 5. Here, the authors detd. the crystal structure of H. pylori I, which had a 1.1 MDa spherical assembly of 12 catalytic units with an outer diam. of ∼160 Å. Under physiol. relevant conditions, the activity of the enzyme remains unaffected down to pH 3. Activity assays under different conditions indicated that the duster of the 12 active sites on the supramol. assembly may be crit. for the survival of the enzyme at low pH. The structure provides a novel example of a mol. assembly adapted for acid resistance that, together with the low Km of the I, is likely to enable the organism to inhabit the hostile niche.
    17. 17
      Phillips, K.; Munster, D. J.; Allardyce, R. A.; Bagshaw, P. F. Antibacterial Action of the Urease Inhibitor Acetohydroxamic acid on Helicobacter pylori. J. Clin. Pathol. 1993, 46, 372– 373,  DOI: 10.1136/jcp.46.4.372
      17
      Antibacterial action of the urease inhibitor acetohydroxamic acid on Helicobacter pylori
      Phillips K; Munster D J; Allardyce R A; Bagshaw P F
      Journal of clinical pathology (1993), 46 (4), 372-3 ISSN:0021-9746.
      The urease inhibitor acetohydroxamic acid (AHA) was assessed for its bacteriostatic and bactericidal effects on Helicobacter pylori. For eight isolates of H pylori, the minimum inhibitory concentration (MIC) was either 200 mg/l or 400 mg/l. Interactions between AHA and antimicrobial drugs used to treat H pylori were also determined. For most isolates AHA reduced the MIC for colloidal bismuth subcitrate (CBS), tetracycline, metronidazole, and amoxicillin. In a few isolates, however, AHA increased the minimum bactericidal concentration (MBC) for these antimicrobial treatments. In vitro AHA is active against H pylori and it interacts with other agents directed against H pylori.
    18. 18
      Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Miletti, S.; Ciurli, S.; Mangani, S. The Complex of Bacillus pasteurii Urease with Acetohydroxamate Anion from X-ray Data at 1.55 Å Resolution. J. Biol. Inorg. Chem. 2000, 5, 110– 118,  DOI: 10.1007/s007750050014
      18
      The complex of Bacillus pasteurii urease with acetohydroxamate anion from x-ray data at 1.55 Å resolution
      Benini, Stefano; Rypniewski, Wojciech R.; Wilson, Keith S.; Miletti, Silvia; Ciurli, Stefano; Mangani, Stefano
      JBIC, Journal of Biological Inorganic Chemistry (2000), 5 (1), 110-118CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)
      The crystal structure of B. pasteurii urease inhibited by acetohydroxamic acid was solved and refined anisotropically using synchrotron x-ray cryogenic diffraction data (1.55 Å resoln.; 99.5% completeness; data redundancy = 26; R-factor = 15.1%, PDB code 4UBP). The 2 Ni2+ ions in the active site were sepd. by a distance of 3.53 Å. The structure clearly showed the binding mode of the inhibitor anion, sym. bridging the 2 Ni2+ ions in the active site through the hydroxamate O atom and chelating 1 Ni2+ ion through the carbonyl O atom. The flexible flap flanking the active site cavity was in the open conformation. The possible implications of the results on structure-based mol. design of new urease inhibitors were discussed.
    19. 19
      Benini, S.; Rypniewski, W.; Wilson, K.; Ciurli, S.; Mangani, S. Structure-based Rationalization of Urease Inhibition by Phosphate: Novel Insights into the Enzyme Mechanism. J. Biol. Inorg. Chem. 2001, 6, 778– 790,  DOI: 10.1007/s007750100254
      19
      Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism
      Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Ciurli, S.; Mangani, S.
      JBIC, Journal of Biological Inorganic Chemistry (2001), 6 (8), 778-790CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)
      The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron x-ray diffraction data from a vitrified crystal (1.85 Å resoln., 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 Å separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom sym. bridges the two metal ions (1.9-2.0 Å), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 Å. The fourth phosphate oxygen is directed into the active site channel. Anal. of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4- anion, and that an addnl. proton is present on the Oδ2 atom of Aspα363, an active site residue involved in Ni coordination through Oδ1. The flexible flap flanking the active site cavity is in the open conformation. Anal. of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.
    20. 20
      Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Mangani, S.; Ciurli, S. Molecular Details of Urease Inhibition by Boric Acid: Insights into the Catalytic Mechanism. J. Am. Chem. Soc. 2004, 126, 3714– 3715,  DOI: 10.1021/ja049618p
      20
      Molecular Details of Urease Inhibition by Boric Acid: Insights into the Catalytic Mechanism
      Benini, Stefano; Rypniewski, Wojciech R.; Wilson, Keith S.; Mangani, Stefano; Ciurli, Stefano
      Journal of the American Chemical Society (2004), 126 (12), 3714-3715CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The structure of the complex of urease, a Ni-contg. metalloenzyme, with boric acid was detd. at 2.10 Å resoln. The complex shows the unprecedented binding mode of the competitive inhibitor to the dinuclear metal center, with the B(OH)3 mol. bridging the Ni ions and leaving in place the bridging hydroxide. Boric acid can be considered a substrate analog of urea, and the structure supports the proposal that the Ni-bridging hydroxide acts as the nucleophile in the enzymic process of urea hydrolysis.
    21. 21
      Benini, S.; Cianci, M.; Mazzei, L.; Ciurli, S. Fluoride inhibition of Sporosarcina pasteurii urease: structure and thermodynamics. J. Biol. Inorg. Chem. 2014, 19, 1243– 1261,  DOI: 10.1007/s00775-014-1182-x
      21
      Fluoride inhibition of Sporosarcina pasteurii urease: structure and thermodynamics
      Benini, Stefano; Cianci, Michele; Mazzei, Luca; Ciurli, Stefano
      JBIC, Journal of Biological Inorganic Chemistry (2014), 19 (8), 1243-1261CODEN: JJBCFA; ISSN:0949-8257. (Springer)
      Urease is a Ni-dependent enzyme and a virulence factor for ureolytic bacterial human pathogens, but it is also necessary to convert urea, the most worldwide-used fertilizer, into forms of nitrogen that can be taken up by crop plants. A strategy to control the activity of urease for medical and agricultural applications is to use enzyme inhibitors. F- is a known urease inhibitor, but the structural basis of its mode of inhibition is still undetd. Here, kinetic studies on the F--induced inhibition of urease from S. pasteurii, a widespread and highly ureolytic soil bacterium, were performed using isothermal titrn. calorimetry and revealed a mixed competitive and uncompetitive mechanism. The pH dependence of the Ki values, investigated in the pH 6.5-8.0 range, revealed a predominant uncompetitive mechanism that increased by increasing the pH, and a lesser competitive inhibition that increased by lowering the pH. Ten crystal structures of the enzyme were independently detd. using 5 crystals of the native form and 5 crystals of the protein crystd. in the presence of F-. The anal. of these structures revealed the presence of 2 F- anions coordinated to the Ni2+ ions in the active site, in terminal and bridging positions. The present study consistently supported an interaction of F- with the Ni centers in the urease active site in which one F- anion competitively bound to Ni2+ proposed to coordinate urea in the initial step of the catalytic mechanism, while another F- anion uncompetitively substituted the Ni2+-bridging hydroxide, blocking its nucleophilic attack on urea.
    22. 22
      Mazzei, L.; Cianci, M.; Musiani, F.; Lente, G.; Palombo, M.; Ciurli, S. Inactivation of Urease by Catechol: Kinetics and Structure. J. inorg. Biochem. 2017, 166, 182– 189,  DOI: 10.1016/j.jinorgbio.2016.11.016
      22
      Inactivation of urease by catechol: Kinetics and structure
      Mazzei, Luca; Cianci, Michele; Musiani, Francesco; Lente, Gabor; Palombo, Marta; Ciurli, Stefano
      Journal of Inorganic Biochemistry (2017), 166 (), 182-189CODEN: JIBIDJ; ISSN:0162-0134. (Elsevier)
      Urease is a Ni(II)-contg. enzyme that catalyzes the hydrolysis of urea to yield NH3 and carbamate at a rate 1015-fold higher than the uncatalyzed reaction. Urease is a virulence factor of several human pathogens, in addn. to decreasing the efficiency of soil org. nitrogen fertilization. Therefore, efficient urease inhibitors are actively sought. Here, the authors describe the characterization of the interaction between urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) with catechol, a model polyphenol. In particular, catechol irreversibly inactivated both SPU and JBU with a complex radical-based autocatalytic multistep mechanism. The crystal structure of the SPU-catechol complex, detd. at 1.50 Å resoln., revealed the structural details of the enzyme inhibition.
    23. 23
      Mazzei, L.; Cianci, M.; Contaldo, U.; Ciurli, S. Insights into Urease Inhibition by N-(n-Butyl) Phosphoric Triamide through an Integrated Structural and Kinetic Approach. J. Agric. Food Chem. 2019, 67, 2127– 2138,  DOI: 10.1021/acs.jafc.8b04791
      23
      Insights into urease inhibition by N-(n-butyl) phosphoric triamide through an integrated structural and kinetic approach
      Mazzei, Luca; Cianci, Michele; Contaldo, Umberto; Ciurli, Stefano
      Journal of Agricultural and Food Chemistry (2019), 67 (8), 2127-2138CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      The nickel-dependent enzyme urease represents a neg. element for the efficiency of soil nitrogen fertilization as well as a virulence factor for a large no. of pathogenic and antibiotic-resistant bacteria. The development of ever more efficient urease inhibitors demands knowledge of their modes of action at the mol. level. N-(n-Butyl)-phosphoric triamide (NBPTO) is the oxo-deriv. of N-(n-butyl)-thiophosphoric triamide (NBPT), which is extensively employed in agriculture to increase the efficiency of urea-based fertilizers. The 1.45 Å resoln. structure of the enzyme-inhibitor complex obtained upon incubation of Sporosarcina pasteurii urease (SPU) with NBPTO shows the presence of diamido phosphoric acid (DAP), generated upon enzymic hydrolysis of NBPTO with the release of Bu amine. DAP is bound in a tridentate binding mode to the two Ni(II) ions in the active site of urease via two O atoms and an amide NH2 group, whereas the second amide group of DAP points away from the metal center into the active-site channel. The mobile flap modulating the size of the active-site cavity is found in a disordered closed-open conformation. A kinetic characterization of the NBPTO-based inhibition of both bacterial (SPU) and plant (Canavalia ensiformis or jack bean, JBU) ureases, carried out by calorimetric measurements, indicates the occurrence of a reversible slow-inhibition mode of action. The latter is characterized by a very small value of the equil. dissocn. const. of the urease-DAP complex caused, in turn, by the large rate const. for the formation of the enzyme-inhibitor complex. The much greater capability of NBPTO to inhibit urease, as compared with that of NBPT, is thus not caused by the presence of a P=O moiety vs. a P=S moiety, as previously suggested, but rather by the readiness of NBPTO to react with urease without the need to convert one of the P-NH2 amide moieties to its P-OH acid deriv., as in the case of NBPT. The latter process is indeed characterized by a very small equil. const. that reduces drastically the concn. of the active form of the inhibitor in the case of NBPT. This indicates that high-efficiency phosphoramide-based urease inhibitors must have at least one O atom bound to the central P atom in order for the mol. to efficiently and rapidly bind to the dinickel center of the enzyme.
    24. 24
      Mazzei, L.; Cianci, M.; Benini, S.; Ciurli, S. The Structure of the Elusive Urease-Urea Complex Unveils the Mechanism of a Paradigmatic Nickel-Dependent Enzyme. Angew. Chem., Int. Ed. 2019, 58, 7415– 7419,  DOI: 10.1002/anie.201903565
      24
      The structure of the elusive urease-urea complex unveils the mechanism of a paradigmatic nickel-dependent enzyme
      Mazzei, Luca; Cianci, Michele; Benini, Stefano; Ciurli, Stefano
      Angewandte Chemie, International Edition (2019), 58 (22), 7415-7419CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Urease, the most efficient enzyme known, contains an essential dinuclear NiII cluster in the active site. It catalyzes the hydrolysis of urea, inducing a rapid pH increase that has neg. effects on human health and agriculture. Thus, the control of urease activity is of utmost importance in medical, pharmaceutical, and agro-environmental applications. All known urease inhibitors are either toxic or inefficient. The development of new and efficient chems. able to inhibit urease relies on the knowledge of all steps of the catalytic mechanism. The short (microseconds) lifetime of the urease-urea complex has hampered the detn. of its structure. The present study uses fluoride to substitute the hydroxide acting as the co-substrate in the reaction, preventing the occurrence of the catalytic steps that follow substrate binding. The 1.42 Å crystal structure of the urease-urea complex, reported here, resolves the enduring debate on the mechanism of this metalloenzyme.
    25. 25
      Karplus, P. A.; Pearson, M. A.; Hausinger, R. P. 70 Years of Crystalline Urease: What Have We Learned?. Acc. Chem. Res. 1997, 30, 330– 337,  DOI: 10.1021/ar960022j
      25
      70 Years of crystalline urease: What have we learned?
      Karplus, P. Andrew; Pearson, Matthew; Hausinger, Robert P.
      Accounts of Chemical Research (1997), 30 (8), 330-337CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review with 60 refs. on the functional and structural characteristics of Klebsiella aerogenes urease. Crystallog. and functional studies of wild-type urease and some site-directed variants suggest a detailed catalytic mechanism which accounts for its kinetic properties and its structure. This mechanism assigns the limiting pKa's of 6.4 and 9.0 to the general acid His-320 and the hydrolytic water, resp. This assignment is such that only a small fraction of the urease mols. are present in the correct protonation state for activity. Obsd. disorder of the solvation in the active site and the comparison of multiple structures provide evidence for suboptimal interactions in the water-filled active site. These suboptimal interactions may lead to an enthalpy-driven free energy gain when this water is released into the bulk solvent upon urea binding. As such, the relaxation of the high-energy state could provide a large amt. of binding energy required for catalysis.
    26. 26
      Pearson, M. A.; Park, I.-S.; Schaller, R. A.; Michel, L. O.; Karplus, P. A.; Hausinger, R. P. Kinetic and Structural Characterization of Urease Active Site Variants. Biochemistry 2000, 39, 8575– 8584,  DOI: 10.1021/bi000613o
      26
      Kinetic and Structural Characterization of Urease Active Site Variants
      Pearson, Matthew A.; Park, Il-Seon; Schaller, Ruth A.; Michel, Linda O.; Karplus, P. Andrew; Hausinger, Robert P.
      Biochemistry (2000), 39 (29), 8575-8584CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >1014-fold the spontaneous rate. To better define the enzyme mechanism, we examd. the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar ∼10-5-fold deficiencies in rates, modest Km changes, and disorders in the peptide flap covering their active sites. H219A urease exhibits 103-fold increased Km over that of native enzyme, whereas the increase is less marked (∼102-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water mols. covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity (∼10-3 that of native enzyme), a small increase in Km, and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a ∼10-4-fold decreased catalytic rate with near-normal pH dependence and an unaffected Km. Phenylglyoxal inactivates the R336Q variant at over half the rate obsd. for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of kcat and kcat/Km for the native enzyme masks a more complex underlying pH dependence involving at least four pKas.
    27. 27
      Dixon, N. E.; Blakeley, R. L.; Zerner, B. Jack Bean Urease (EC 3.5.1.5). III. The Involvement of Active-site Nickel Ion in Inhibition by b-Mercaptoethanol, Phosphoramidate, and Fluoride. Can. J. Biochem. 1980, 58, 481– 488,  DOI: 10.1139/o80-064
      27
      Jack bean urease (EC 3.5.1.5). III. The involvement of active-site nickel ion in inhibition by β-mercaptoethanol, phosphoramidate, and fluoride
      Dixon, Nicholas E.; Blakeley, Robert L.; Zerner, Burt
      Canadian Journal of Biochemistry (1980), 58 (6), 481-8CODEN: CJBIAE; ISSN:0008-4018.
      Interaction of β-mercaptoethanol with urease produces large, rapid, and fully reversible spectral changes in that part of the electronic absorption spectrum which is assocd. with the tightly bound Ni2+. The spectrophotometrically detd. value of the dissocn. const. of the β-mercaptoethanol-urease complex (0.95 mM at pH 7.12 and 25°) is in agreement with the Ki (0.72 mM) for β-mercaptoethanol acting as a competitive inhibitor in the hydrolysis of urea. This constitutes direct evidence that the Ni2+ in jack bean urease is at the active site. Inhibition of urease by phosphoramidate is slowly achieved and slowly reversed, and upon reactivation of the isolated phosphoramidate-urease complex, phosphoramidate is regenerated in good yield. Spectrophotometric expts. indicate that phosphoramidate binds to Ni2+ in urease. Competition with β-mercaptoethanol was used to det. a dissocn. const. (1.23 mM at pH 7.12 and 25°) for a F--urease complex in which F- also coordinates with an active-site Ni2+. Kinetic evidence is presented which indicates that in the presence of urea, a ternary complex (F-urea-urease) is formed.
    28. 28
      Pelmenschikov, V.; Siegbahn, P. E. Nickel Superoxide Dismutase Reaction Mechanism Studied by Hybrid Density Functional Methods. J. Am. Chem. Soc. 2006, 128, 7466– 7475,  DOI: 10.1021/ja053665f
      28
      Nickel Superoxide Dismutase Reaction Mechanism Studied by Hybrid Density Functional Methods
      Pelmenschikov, Vladimir; Siegbahn, Per E. M.
      Journal of the American Chemical Society (2006), 128 (23), 7466-7475CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The reaction mechanism for the disproportionation of the toxic superoxide radical to mol. oxygen and hydrogen peroxide by the nickel-dependent superoxide dismutase (NiSOD) has been studied using the B3LYP hybrid DFT method. Based on the recent x-ray structures of the enzyme in the resting oxidized Ni(III) and x-ray-reduced Ni(II) states, the model investigated includes the backbone spacer of six residues (sequence nos. 1-6) as a structural framework. The side chains of residues His-1, Cys-2, and Cys-6, which are essential for nickel binding and catalysis, were modeled explicitly. The catalytic cycle consists of two half-reactions, each initiated by the successive substrate approach to the metal center. The two protons necessary for the dismutation are postulated to be delivered concertedly with the superoxide radical anions. The first (reductive) phase involves Ni(III) redn. to Ni(II), and the second (oxidative) phase involves the metal reoxidn. back to its resting state. The Cys-2 thiolate sulfur serves as a transient protonation site in the interim between the two half-reactions, allowing for the dioxygen and hydrogen peroxide mols. to be released in the reductive and oxidative phases, resp. The His-1 side chain nitrogen and backbone amides of the active site channel are shown to be less favorable transient proton locations, as compared to the Cys-2 sulfur. Comparisons are made to the Cu- and Zn-dependent SOD, studied previously using similar models.
    29. 29
      Sparta, M.; Valdez, C. E.; Alexandrova, A. N. Metal-dependent Activity of Fe and Ni Acireductone Dioxygenases: How Two Electrons Reroute the Catalytic Pathway. J. Mol. Biol. 2013, 425, 3007– 3018,  DOI: 10.1016/j.jmb.2013.05.001
      29
      Metal-Dependent Activity of Fe and Ni Acireductone Dioxygenases: How Two Electrons Reroute the Catalytic Pathway
      Sparta, Manuel; Valdez, Crystal E.; Alexandrova, Anastassia N.
      Journal of Molecular Biology (2013), 425 (16), 3007-3018CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Two virtually identical acireductone dioxygenases, ARD and ARD', catalyze completely different oxidn. reactions of the same substrate, 1,2-dihydroxy-3-keto-5-(methylthio)pentene, depending exclusively on the nature of the bound metal. Fe2+-dependent ARD' produces the α-keto acid precursor of methionine and formate and allows for the recycling of methionine in cells. Ni2+-dependent ARD instead produces methylthiopropionate, CO, and formate, and exits the methionine salvage cycle. This mechanistic difference has not been understood to date but has been speculated to be due to the difference in coordination of the substrate to Fe2+ vs. Ni2+: forming a five-membered ring vs. a six-membered ring, resp., thus exposing different carbon atoms for the attack by O2. Here, using mixed quantum-classical mol. dynamics simulations followed by the d. functional theory mechanistic investigation, we show that contrary to the old hypothesis, both metals preferentially bind the substrate as a six-membered ring, exposing the exact same sites to the attack by O2. It is the electronic properties of the metals that are then responsible for the system following different reaction paths, to yield the resp. products. We fully explain the puzzling metal-induced difference in functionality between ARD and ARD' and, in particular, propose a new mechanism for ARD'. All results are in agreement with available isotopic substitution and other exptl. data.
    30. 30
      Wang, W.-J.; Wei, W.-J.; Liao, R.-Z. Deciphering the Chemoselectivity of Nickel-dependent Quercetin 2, 4-Dioxygenase. Phys. Chem. Chem. Phys. 2018, 20, 15784– 15794,  DOI: 10.1039/C8CP02683A
      30
      Deciphering the chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase
      Wang, Wen-Juan; Wei, Wen-Jie; Liao, Rong-Zhen
      Physical Chemistry Chemical Physics (2018), 20 (23), 15784-15794CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
      The reaction mechanism and chemoselectivity of nickel-dependent quercetin 2,4-dioxygenase (2,4-QueD) have been investigated using the QM/MM approach. The protonation state of the Glu74 residue, a first-shell ligand of Ni, has been considered to be either neutral or deprotonated. QM/MM calcns. predict that Glu74 must be deprotonated to rationalize the chemoselectivity and steer the 2,4-dioxygenolytic cleavage of quercetin, which harvests the exptl.-obsd. product, 2-protocatechuoylphloroglucinol carboxylic acid, coupled with the release of carbon monoxide. If the enzyme has a neutral Glu74 residue, the undesired 2,3-dioxygenolytic cleavage of quercetin becomes the dominant pathway, leading to the formation of α-keto acid. The calcns. suggest that the reaction takes place via three major steps: (1) attack of the superoxide on the C2 of the substrate pyrone ring to generate a NiII-peroxide intermediate; (2) formation of the second C-O bond between C4 and the peroxide to produce a peroxide bridge; (3) simultaneous cleavage of the C2-C3, C3-C4, and O1-O2 bonds with the formation of 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The third step was found to be rate-limiting, with a barrier of 17.4 kcal mol-1, which is in very good agreement with the exptl. kinetic data. For the second C-O bond formation, an alternative pathway is that the peroxide attacks the C3 of the substrate pyrone ring, leading to the formation of a four-membered ring intermediate, which then undergoes concerted C2-C3 and O1-O2 bond cleavages to produce an α-keto acid. This pathway is assocd. with a barrier of 30.6 kcal mol-1, which is much higher than the major pathway. When Glu74 is protonated, the 2,3-dioxygenolytic pathway, however, has a lower barrier (21.8 kcal mol-1) than the 2,4-dioxygenolytic pathway.
    31. 31
      Siegbahn, P. E.; Chen, S.-L.; Liao, R.-Z. Theoretical Studies of Nickel-dependent Enzymes. Inorganics 2019, 7, 95,  DOI: 10.3390/inorganics7080095
      31
      Theoretical studies of nickel-dependent enzymes
      Siegbahn, Per E. M.; Chen, Shi-Lu; Liao, Rong-Zhen
      Inorganics (2019), 7 (8), 95CODEN: INORCW; ISSN:2304-6740. (MDPI AG)
      The advancements of quantum chem. methods and computer power allow detailed mechanistic investigations of metalloenzymes. In particular, both quantum chem. cluster and combined QM/MM approaches have been used, which have been proven to successfully complement exptl. studies. This review starts with a brief introduction of nickel-dependent enzymes and then summarizes theor. studies on the reaction mechanisms of these enzymes, including NiFe hydrogenase, methyl-coenzyme M reductase, nickel CO dehydrogenase, acetyl CoA synthase, acireductone dioxygenase, quercetin 2,4-dioxygenase, urease, lactate racemase, and superoxide dismutase.
    32. 32
      Suárez, D.; Díaz, N.; Merz, K. M. Ureases: Quantum Chemical Calculations on Cluster Models. J. Am. Chem. Soc. 2003, 125, 15324– 15337,  DOI: 10.1021/ja030145g
      32
      Ureases: Quantum Chemical Calculations on Cluster Models
      Suarez, Dimas; Diaz, Natalia; Merz, Kenneth M., Jr.
      Journal of the American Chemical Society (2003), 125 (50), 15324-15337CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Herein, the authors present results from a computational study of dinickel complexes that are relevant to the catalytic hydrolysis of urea exerted by the urease enzymes. The B3LYP d. functional is used to characterize the equil. geometry, electronic and magnetic properties, and energies for a series of realistic complexes modeling the active site of ureases. The anal. of the theor. results gives new insight into the structure, substrate binding, and catalytic mechanism. The water bridge between the two Ni(II) ions obsd. in the crystallog. structures of the ureases was assigned to a hydroxide bridge in agreement with the obsd. small antiferromagnetic coupling. Both monodentate and bidentate urea-bound complexes, in which urea had favorable orientations for catalysis, were characterized. Finally, two reaction mechanisms were investigated starting from the monodentate and bidentate urea-bound complexes, resp. Both a Ni1···Ni2 bridging hydroxide and a Ni2-bound water mol. play crucial roles in the two mechanisms.
    33. 33
      Estiu, G.; Merz, K. M. Competitive Hydrolytic and Elimination Mechanisms in the Urease Catalyzed Decomposition of Urea. J. Phys. Chem. B 2007, 111, 10263– 10274,  DOI: 10.1021/jp072323o
      33
      Competitive Hydrolytic and Elimination Mechanisms in the Urease Catalyzed Decomposition of Urea
      Estiu, Guillermina; Merz, Kenneth M., Jr.
      Journal of Physical Chemistry B (2007), 111 (34), 10263-10274CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      We present a high-level quantum chem. study of possible elimination reaction mechanisms assocd. with the catalytic decompn. of urea at the binuclear nickel active site cluster of urease. Stable intermediates and transition state structures have been identified along several possible reaction pathways. The computed results are compared with those reported by Suarez et al. for the hydrolytic catalyzed decompn. On the basis of these comparative studies, we propose a monodentate coordination of urea in the active site from which both the elimination and hydrolytic pathways can decomp. urea into CO2 and NH3. This observation is counter to what has been exptl. suggested based on the exogenous observation of carbamic acid (the reaction product from the hydrolysis pathway). However, this does not address what has occurred at the active site of urease prior to product release. On the basis of our computed results, the observation that urea prefers the elimination channel in aq. soln. and on the observation of Lippard and co-workers of an elimination reaction channel in a urease biomimetic model, we propose that the elimination channel needs to be re-examd. as a viable reaction channel in urease.
    34. 34
      Carlsson, H.; Nordlander, E. Computational Modeling of the Mechanism of Urease. Bioinorg. Chem. Appl. 2010, 2010, 364891,  DOI: 10.1155/2010/364891
      There is no corresponding record for this reference.
    35. 35
      Barrios, A. M.; Lippard, S. J. Interaction of Urea with a Hydroxide-Bridged Dinuclear Nickel Center: An Alternative Model for the Mechanism of Urease. J. Am. Chem. Soc. 2000, 122, 9172– 9177,  DOI: 10.1021/ja000202v
      35
      Interaction of Urea with a Hydroxide-Bridged Dinuclear Nickel Center: An Alternative Model for the Mechanism of Urease
      Barrios, Amy M.; Lippard, Stephen J.
      Journal of the American Chemical Society (2000), 122 (38), 9172-9177CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      A hydroxide-bridged dinuclear nickel complex with a urea mol. linking the two metal ions through its carbonyl oxygen atom has been prepd. as a model for the metalloenzyme urease. This complex, [Ni2(μ-OH)(μ-urea)(bdptz)(urea)(CH3CN)](ClO4)3, where bdptz is the dinucleating ligand 1,4-bis(2,2'-dipyridylmethyl)phthalazine, effects the hydrolysis of urea upon heating in a two-step reaction. In the first step, a mol. of ammonia is eliminated from urea with concomitant prodn. of cyanate, the first-order rate const. in acetonitrile being (7.7 ± 0.5) × 10-4 h-1. This reaction is at least 500 times faster than the spontaneous decompn. of urea under the same conditions. When the cyanate-contg. product is further heated in the presence of water, the cyanate is hydrolyzed with a second-order rate const. of (9.5 ± 1) × 10-4 M-1 h-1. Reaction of [Ni2(μ-OH)(μ-urea)(bdptz)(urea)(CH3CN)](ClO4)3 in 50% aq. acetonitrile afforded ammonia with no appreciable buildup of the cyanate-contg. species. A possible analog of the cyanate-contg. product, [Ni2(μ-OH)(μ-H2O)(bdptz)(μ-OCN)]2(OTs)4, was independently synthesized and structurally characterized. These results establish the precedence for hydrolysis of urea via a cyanate intermediate as an alternative mechanism for the urease-catalyzed hydrolysis of urea.
    36. 36
      Senn, H. M.; Thiel, W. QM/MM Methods for Biomolecular Systems. Angew. Chem., Int. Ed. 2009, 48, 1198– 1229,  DOI: 10.1002/anie.200802019
      36
      QM/MM methods for biomolecular systems
      Senn, Hans Martin; Thiel, Walter
      Angewandte Chemie, International Edition (2009), 48 (7), 1198-1229CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Combined quantum-mechanics/mol.-mechanics (QM/MM) approaches have become the method of choice for modeling reactions in biomol. systems. Quantum-mech. (QM) methods are required for describing chem. reactions and other electronic processes, such as charge transfer or electronic excitation. However, QM methods are restricted to systems of up to a few hundred atoms. However, the size and conformational complexity of biopolymers calls for methods capable of treating up to several 100,000 atoms and allowing for simulations over time scales of tens of nanoseconds. This is achieved by highly efficient, force-field-based mol. mechanics (MM) methods. Thus to model large biomols. the logical approach is to combine the two techniques and, to use a QM method for the chem. active region (e.g., substrates and co-factors in an enzymic reaction) and an MM treatment for the surroundings (e.g., protein and solvent). The resulting schemes are commonly referred to as combined or hybrid QM/MM methods. They enable the modeling of reactive biomol. systems at a reasonable computational effort while providing the necessary accuracy.
    37. 37
      Ahmadi, S.; Barrios Herrera, L.; Chehelamirani, M.; Hostaš, J.; Jalife, S.; Salahub, D. R. Multiscale Modeling of Enzymes: QM-cluster, QM/MM, and QM/MM/MD: A Tutorial Review. Int. J. Quantum Chem. 2018, 118, e25558,  DOI: 10.1002/qua.25558
      There is no corresponding record for this reference.
    38. 38
      Neese, F. The ORCA Program System. WIREs Comput. Mol. Sci. 2012, 2, 73– 78,  DOI: 10.1002/wcms.81
      38
      The ORCA program system
      Neese, Frank
      Wiley Interdisciplinary Reviews: Computational Molecular Science (2012), 2 (1), 73-78CODEN: WIRCAH; ISSN:1759-0884. (Wiley-Blackwell)
      A review. ORCA is a general-purpose quantum chem. program package that features virtually all modern electronic structure methods (d. functional theory, many-body perturbation and coupled cluster theories, and multireference and semiempirical methods). It is designed with the aim of generality, extendibility, efficiency, and user friendliness. Its main field of application is larger mols., transition metal complexes, and their spectroscopic properties. ORCA uses std. Gaussian basis functions and is fully parallelized. The article provides an overview of its current possibilities and documents its efficiency.
    39. 39
      Becke, A. D. Density-functional Exchange-energy Approximation with Correct Asymptotic Behavior. Phys. Rev. A 1988, 38, 3098– 3100,  DOI: 10.1103/PhysRevA.38.3098
      39
      Density-functional exchange-energy approximation with correct asymptotic behavior
      Becke, A. D.
      Physical Review A: Atomic, Molecular, and Optical Physics (1988), 38 (6), 3098-100CODEN: PLRAAN; ISSN:0556-2791.
      Current gradient-cor. d.-functional approxns. for the exchange energies of at. and mol. systems fail to reproduce the correct 1/r asymptotic behavior of the exchange-energy d. A gradient-cor. exchange-energy functional is given with the proper asymptotic limit. This functional, contg. only one parameter, fits the exact Hartree-Fock exchange energies of a wide variety of at. systems with remarkable accuracy, surpassing the performance of previous functionals contg. two parameters or more.
    40. 40
      Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-energy Formula into a Functional of the Electron Density. Phys. Rev. B 1988, 37, 785– 789,  DOI: 10.1103/PhysRevB.37.785
      40
      Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density
      Lee, Chengteh; Yang, Weitao; Parr, Robert G.
      Physical Review B: Condensed Matter and Materials Physics (1988), 37 (2), 785-9CODEN: PRBMDO; ISSN:0163-1829.
      A correlation-energy formula due to R. Colle and D. Salvetti (1975), in which the correlation energy d. is expressed in terms of the electron d. and a Laplacian of the 2nd-order Hartree-Fock d. matrix, is restated as a formula involving the d. and local kinetic-energy d. On insertion of gradient expansions for the local kinetic-energy d., d.-functional formulas for the correlation energy and correlation potential are then obtained. Through numerical calcns. on a no. of atoms, pos. ions, and mols., of both open- and closed-shell type, it is demonstrated that these formulas, like the original Colle-Salvetti formulas, give correlation energies within a few percent.
    41. 41
      Becke, A. D. Density-functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. phys. 1993, 98, 5648– 5652,  DOI: 10.1063/1.464913
      41
      Density-functional thermochemistry. III. The role of exact exchange
      Becke, Axel D.
      Journal of Chemical Physics (1993), 98 (7), 5648-52CODEN: JCPSA6; ISSN:0021-9606.
      Despite the remarkable thermochem. accuracy of Kohn-Sham d.-functional theories with gradient corrections for exchange-correlation, the author believes that further improvements are unlikely unless exact-exchange information is considered. Arguments to support this view are presented, and a semiempirical exchange-correlation functional (contg. local-spin-d., gradient, and exact-exchange terms) is tested for 56 atomization energies, 42 ionization potentials, 8 proton affinities, and 10 total at. energies of first- and second-row systems. This functional performs better than previous functionals with gradient corrections only, and fits expt. atomization energies with an impressively small av. abs. deviation of 2.4 kcal/mol.
    42. 42
      Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J. Ab Initio Calculation of Vibrational Absorption and Circular Dichroism Spectra Using Density Functional Force Fields. J. Phys. Chem. 1994, 98, 11623– 11627,  DOI: 10.1021/j100096a001
      42
      Ab Initio Calculation of Vibrational Absorption and Circular Dichroism Spectra Using Density Functional Force Fields
      Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J.
      Journal of Physical Chemistry (1994), 98 (45), 11623-7CODEN: JPCHAX; ISSN:0022-3654.
      The unpolarized absorption and CD spectra of the fundamental vibrational transitions of the chiral mol. 4-methyl-2-oxetanone are calcd. ab initio. Harmonic force fields are obtained using d. functional theory (DFT), MP2 and SCF methodologies, and a [5s4p2d/3s2p] (TZ2P) basis set. DFT calcns. use the LSDA, BLYP, and Becke3LYP (B3LYP) d. functionals. Mid-IR spectra predicted using LSDA, BLYP, and B3LYP force fields are of significantly different quality, the B3LYP force field yielding spectra in clearly superior, and overall excellent, agreement with expt. The MP2 force field yields spectra in slightly worse agreement with expt. than the B3LYP force field. The SCF force field yields spectra in poor agreement with expt. The basis set dependence of B3LYP force fields is also explored: the 6-31G* and TZ2P basis sets give very similar results while the 3-21G basis set yields spectra in substantially worse agreement with expt.
    43. 43
      Neese, F.; Wennmohs, F.; Hansen, A.; Becker, U. Efficient, Approximate and Parallel Hartree-Fock and Hybrid DFT Calculations. A ‘Chain-of-Spheres’ Algorithm for the Hartree-Fock Exchange. Chem. Phys. 2009, 356, 98– 109,  DOI: 10.1016/j.chemphys.2008.10.036
      43
      Efficient, approximate and parallel Hartree-Fock and hybrid DFT calculations. A 'chain-of-spheres' algorithm for the Hartree-Fock exchange
      Neese, Frank; Wennmohs, Frank; Hansen, Andreas; Becker, Ute
      Chemical Physics (2009), 356 (1-3), 98-109CODEN: CMPHC2; ISSN:0301-0104. (Elsevier B.V.)
      In this paper, the possibility is explored to speed up Hartree-Fock and hybrid d. functional calcns. by forming the Coulomb and exchange parts of the Fock matrix by different approxns. For the Coulomb part the previously introduced Split-RI-J variant of the well-known d. fitting' approxn. is used. The exchange part is formed by semi-numerical integration techniques that are closely related to Friesner's pioneering pseudo-spectral approach. Our potentially linear scaling realization of this algorithm is called the 'chain-of-spheres exchange' (COSX). A combination of semi-numerical integration and d. fitting is also proposed. Both Split-RI-J and COSX scale very well with the highest angular momentum in the basis sets. It is shown that for extended basis sets speed-ups of up to two orders of magnitude compared to traditional implementations can be obtained in this way. Total energies are reproduced with an av. error of <0.3 kcal/mol as detd. from extended test calcns. with various basis sets on a set of 26 mols. with 20-200 atoms and up to 2000 basis functions. Reaction energies agree to within 0.2 kcal/mol (Hartree-Fock) or 0.05 kcal/mol (hybrid DFT) with the canonical values. The COSX algorithm parallelizes with a speedup of 8.6 obsd. for 10 processes. Min. energy geometries differ by less than 0.3 pm in the bond distances and 0.5° in the bond angles from their canonical values. These developments enable highly efficient and accurate SCF calcns. including nonlocal Hartree-Fock exchange for large mols. In combination with the RI-MP2 method and large basis sets, second-order many body perturbation energies can be obtained for medium sized mols. with unprecedented efficiency. The algorithms are implemented into the ORCA electronic structure system.

      Moving Frontiers in Quantum Chemistry.

    44. 44
      Weigend, F.; Ahlrichs, R. Balanced Basis Sets of Split Valence, Triple Zeta Valence and Quadruple Zeta Valence Quality for H to Rn: Design and Assessment of Accuracy. Phys. Chem. Chem. Phys. 2005, 7, 3297– 3305,  DOI: 10.1039/b508541a
      44
      Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy
      Weigend, Florian; Ahlrichs, Reinhart
      Physical Chemistry Chemical Physics (2005), 7 (18), 3297-3305CODEN: PPCPFQ; ISSN:1463-9076. (Royal Society of Chemistry)
      Gaussian basis sets of quadruple zeta valence quality for Rb-Rn are presented, as well as bases of split valence and triple zeta valence quality for H-Rn. The latter were obtained by (partly) modifying bases developed previously. A large set of more than 300 mols. representing (nearly) all elements-except lanthanides-in their common oxidn. states was used to assess the quality of the bases all across the periodic table. Quantities investigated were atomization energies, dipole moments and structure parameters for Hartree-Fock, d. functional theory and correlated methods, for which we had chosen Moller-Plesset perturbation theory as an example. Finally recommendations are given which type of basis set is used best for a certain level of theory and a desired quality of results.
    45. 45
      Breneman, C. M.; Wiberg, K. B. Determining Atom-centered Monopoles from Molecular Electrostatic Potentials. The Need for High Sampling Density in Formamide Conformational Analysis. J. Comput. Chem. 1990, 11, 361– 373,  DOI: 10.1002/jcc.540110311
      45
      Determining atom-centered monopoles from molecular electrostatic potentials. The need for high sampling density in formamide conformational analysis
      Breneman, Curt M.; Wiberg, Kenneth B.
      Journal of Computational Chemistry (1990), 11 (3), 361-73CODEN: JCCHDD; ISSN:0192-8651.
      An improved method for computing potential-derived charges is described which is based upon the CHELP program available from QCPE. This approach (CHELPG) is shown to be considerably less dependent upon mol. orientation than the original CHELP program. In the second part of this work, the CHELPG point selection algorithm was used to analyze the changes in the potential-derived charges in formamide during rotation about the C-N bond. In order to achieve a level of rotational invariance less than 10% of the magnitude of the electronic effects studied, an equally-spaced array of points 0.3 Å apart was required. Points found to be greater than 2.8 Å from any nucleus were eliminated, along with all points contained within the defined VDW distances from each of the atoms. The results are compared to those obtained by using CHELP. Even when large nos. of points (ca. 3000) were sampled using the CHELP selection routine, the results did not indicate a satisfactory level of rotational invariance. On the basis of these results, the original CHELP program was inadequate for analyzing internal rotations.
    46. 46
      Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.; Mackerell, A. D., Jr. CHARMM General Force Field: A Force Field for Drug-like Molecules Compatible with the CHARMM All-atom Additive Biological Force Fields. J. Comput. Chem. 2010, 31, 671– 690,  DOI: 10.1002/jcc.21367
      46
      CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields
      Vanommeslaeghe, K.; Hatcher, E.; Acharya, C.; Kundu, S.; Zhong, S.; Shim, J.; Darian, E.; Guvench, O.; Lopes, P.; Vorobyov, I.; Mackerell, A. D., Jr.
      Journal of Computational Chemistry (2010), 31 (4), 671-690CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      The widely used CHARMM additive all-atom force field includes parameters for proteins, nucleic acids, lipids, and carbohydrates. In the present article, an extension of the CHARMM force field to drug-like mols. is presented. The resulting CHARMM General Force Field (CGenFF) covers a wide range of chem. groups present in biomols. and drug-like mols., including a large no. of heterocyclic scaffolds. The parametrization philosophy behind the force field focuses on quality at the expense of transferability, with the implementation concg. on an extensible force field. Statistics related to the quality of the parametrization with a focus on exptl. validation are presented. Addnl., the parametrization procedure, described fully in the present article in the context of the model systems, pyrrolidine, and 3-phenoxymethyl-pyrrolidine will allow users to readily extend the force field to chem. groups that are not explicitly covered in the force field as well as add functional groups to and link together mols. already available in the force field. CGenFF thus makes it possible to perform "all-CHARMM" simulations on drug-target interactions thereby extending the utility of CHARMM force fields to medicinally relevant systems. © 2009 Wiley Periodicals, Inc.J Comput Chem, 2010.
    47. 47
      Li, P.; Roberts, B. P.; Chakravorty, D. K.; Merz, K. M., Jr. Rational Design of Particle Mesh Ewald Compatible Lennard-Jones Parameters for + 2 Metal Cations in Explicit Solvent. J. Chem. Theory Comput. 2013, 9, 2733– 2748,  DOI: 10.1021/ct400146w
      47
      Rational Design of Particle Mesh Ewald Compatible Lennard-Jones Parameters for +2 Metal Cations in Explicit Solvent
      Li, Pengfei; Roberts, Benjamin P.; Chakravorty, Dhruva K.; Merz, Kenneth M.
      Journal of Chemical Theory and Computation (2013), 9 (6), 2733-2748CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Metal ions play significant roles in biol. systems. Accurate mol. dynamics (MD) simulations on these systems require a validated set of parameters. Although there are more detailed ways to model metal ions, the nonbonded model, which employs a 12-6 Lennard-Jones (LJ) term plus an electrostatic potential, is still widely used in MD simulations today due to its simple form. However, LJ parameters have limited transferability due to different combining rules, various water models, and diverse simulation methods. Recently, simulations employing a Particle Mesh Ewald (PME) treatment for long-range electrostatics have become more and more popular owing to their speed and accuracy. In the present work, we have systematically designed LJ parameters for 24 +2 metal (M(II)) cations to reproduce different exptl. properties appropriate for the Lorentz-Berthelot combining rules and PME simulations. We began by testing the transferability of currently available M(II) ion LJ parameters. The results showed that there are differences between simulations employing Ewald summation with other simulation methods and that it was necessary to design new parameters specific for PME based simulations. Employing the thermodn. integration (TI) method and performing periodic boundary MD simulations employing PME, allowed for a systematic investigation of the LJ parameter space. Hydration free energies (HFEs), the ion-oxygen distance in the first solvation shell (IOD), and coordination nos. (CNs) were obtained for various combinations of the parameters of the LJ potential for four widely used water models (TIP3P, SPC/E, TIP4P, and TIP4PEW). Results showed that the three simulated properties were highly correlated. Meanwhile, M(II) ions with the same parameters in different water models produce remarkably different HFEs but similar structural properties. It is difficult to reproduce various exptl. values simultaneously because the nonbonded model underestimates the interaction between the metal ions and water mols. at short-range. Moreover, the extent of underestimation increases successively for the TIP3P, SPC/E, TIP4PEW, and TIP4P water models. Nonetheless, we fitted a curve to describe the relationship between ε (the well depth) and radius (Rmin/2) from exptl. data on noble gases to facilitate the generation of the best possible compromise models. Hence, by targeting different exptl. values, we developed three sets of parameters for M(II) cations for three different water models (TIP3P, SPC/E, and TIP4PEW). These parameters we feel represent the best possible compromise that can be achieved using the nonbonded model for the ions in combination with simple water models. From a computational uncertainty anal. we est. that the uncertainty in our computed HFEs is on the order of ±1 kcal/mol. Further improvements will require more advanced nonbonded models likely with inclusion of polarization.
    48. 48
      Lee, J.; Cheng, X.; Swails, J. M.; Yeom, M. S.; Eastman, P. K.; Lemkul, J. A.; Wei, S.; Buckner, J.; Jeong, J. C.; Qi, Y. CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field. J. Chem. Theory Comput. 2016, 12, 405– 413,  DOI: 10.1021/acs.jctc.5b00935
      48
      CHARMM-GUI Input Generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM Simulations Using the CHARMM36 Additive Force Field
      Lee, Jumin; Cheng, Xi; Swails, Jason M.; Yeom, Min Sun; Eastman, Peter K.; Lemkul, Justin A.; Wei, Shuai; Buckner, Joshua; Jeong, Jong Cheol; Qi, Yifei; Jo, Sunhwan; Pande, Vijay S.; Case, David A.; Brooks, Charles L.; MacKerell, Alexander D.; Klauda, Jeffery B.; Im, Wonpil
      Journal of Chemical Theory and Computation (2016), 12 (1), 405-413CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      Proper treatment of nonbonded interactions is essential for the accuracy of mol. dynamics (MD) simulations, esp. in studies of lipid bilayers. The use of the CHARMM36 force field (C36 FF) in different MD simulation programs can result in disagreements with published simulations performed with CHARMM due to differences in the protocols used to treat the long-range and 1-4 nonbonded interactions. In this study, we systematically test the use of the C36 lipid FF in NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM. A wide range of Lennard-Jones (LJ) cutoff schemes and integrator algorithms were tested to find the optimal simulation protocol to best match bilayer properties of six lipids with varying acyl chain satn. and head groups. MD simulations of a 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) bilayer were used to obtain the optimal protocol for each program. MD simulations with all programs were found to reasonably match the DPPC bilayer properties (surface area per lipid, chain order parameters, and area compressibility modulus) obtained using the std. protocol used in CHARMM as well as from expts. The optimal simulation protocol was then applied to the other five lipid simulations and resulted in excellent agreement between results from most simulation programs as well as with exptl. data. AMBER compared least favorably with the expected membrane properties, which appears to be due to its use of the hard-truncation in the LJ potential vs. a force-based switching function used to smooth the LJ potential as it approaches the cutoff distance. The optimal simulation protocol for each program has been implemented in CHARMM-GUI. This protocol is expected to be applicable to the remainder of the additive C36 FF including the proteins, nucleic acids, carbohydrates, and small mols.
    49. 49
      Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graph. 1996, 14, 33– 38,  DOI: 10.1016/0263-7855(96)00018-5
      49
      VDM: visual molecular dynamics
      Humphrey, William; Dalke, Andrew; Schulten, Klaus
      Journal of Molecular Graphics (1996), 14 (1), 33-8, plates, 27-28CODEN: JMGRDV; ISSN:0263-7855. (Elsevier)
      VMD is a mol. graphics program designed for the display and anal. of mol. assemblies, in particular, biopolymers such as proteins and nucleic acids. VMD can simultaneously display any no. of structures using a wide variety of rendering styles and coloring methods. Mols. are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resoln. raster images of displayed mols. may be produced by generating input scripts for use by a no. of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate mol. dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biol., which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs, VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
    50. 50
      Park, I.-S.; Hausinger, R. P. Site-Directed Mutagenesis of Klebsiella aerogenes Urease: Identification of Histidine Residues that Appear to Function in Nickel Ligation, Substrate Binding, and Catalysis. Protein Sci. 1993, 2, 1034– 1041,  DOI: 10.1002/pro.5560020616
      50
      Site-directed mutagenesis of Klebsiella aerogenes urease: Identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis
      Park, Il Seon; Hausinger, Robert P.
      Protein Science (1993), 2 (6), 1034-41CODEN: PRCIEI; ISSN:0961-8368.
      Comparison of six urease sequences revealed the presence of 10 conserved histidine residues (H96 in the γ subunit, H39 and H41 in β, and H134, H136, H219, H246, H312, H320, and H321 in the α subunit of the K. aerogenes enzyme). Each of these residues in K. aerogenes urease was substituted with alanine by site-directed mutagenesis, and the mutant proteins were purified and characterized in order to identify essential histidine residues and assign their roles. The γH96A, βH39A, βH41A, αH312A, and αH321A mutant proteins possess activities and nickel contents similar to wild-type enzyme, suggesting that these residues are not essential for substrate binding, catalysis, or metal binding. In contrast, the αH134A, αH136A, and αH246A proteins exhibit no detectable activity and possess 53%, 6%, and 21% of the nickel content of wild-type enzyme. These results are consistent with αH134, αH136, and αH246 functioning as nickel ligands. The αH219A protein is active and has nickel (∼1.9% and ∼80%, resp., when compared to wild-type protein) but exhibits a very high Km value (1,100 mM compared to 2.3 mM for the wild-type enzyme). These results are compatible with αH219 having some role in facilitating substrate binding. Finally, the αH320A protein (Km = 8.3 mM) only displays ∼0.003% of the wild-type enzyme activity, despite having a normal nickel content. Unlike the wild-type and αH219A ureases, this mutant protein was not inactivated by diethylpyrocarbonate (DEP), consistent with αH320 being the DEP-reactive general base that facilitates catalysis.
    51. 51
      Nelson, M. T.; Humphrey, W.; Gursoy, A.; Dalke, A.; Kalé, L. V.; Skeel, R. D.; Schulten, K. NAMD: A Parallel, Object-Oriented Molecular Dynamics Program. Int. J. High Perform. Comput. Appl. 1996, 10, 251– 268,  DOI: 10.1177/109434209601000401
      There is no corresponding record for this reference.
    52. 52
      Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald: An N·log (N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089– 10092,  DOI: 10.1063/1.464397
      52
      Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems
      Darden, Tom; York, Darrin; Pedersen, Lee
      Journal of Chemical Physics (1993), 98 (12), 10089-92CODEN: JCPSA6; ISSN:0021-9606.
      An N·log(N) method for evaluating electrostatic energies and forces of large periodic systems is presented. The method is based on interpolation of the reciprocal space Ewald sums and evaluation of the resulting convolution using fast Fourier transforms. Timings and accuracies are presented for three large cryst. ionic systems.
    53. 53
      Huang, J.; MacKerell, A. D., Jr CHARMM36 All-atom Additive Protein Force Field: Validation Based on Comparison to NMR Data. J. Comput. Chem. 2013, 34, 2135– 2145,  DOI: 10.1002/jcc.23354
      53
      CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data
      Huang, Jing; MacKerell, Alexander D. Jr
      Journal of Computational Chemistry (2013), 34 (25), 2135-2145CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)
      Protein structure and dynamics can be characterized on the atomistic level with both NMR expts. and mol. dynamics (MD) simulations. Here, the authors quantify the ability of the recently presented CHARMM36 (C36) force field (FF) to reproduce various NMR observables using MD simulations. The studied NMR properties include backbone scalar couplings across hydrogen bonds, residual dipolar couplings (RDCs) and relaxation order parameter, as well as scalar couplings, RDCs, and order parameters for side-chain amino- and methyl-contg. groups. The C36 FF leads to better correlation with exptl. data compared to the CHARMM22/CMAP FF and suggest using C36 in protein simulations. Although both CHARMM FFs contains the same nonbond parameters, the authors' results show how the changes in the internal parameters assocd. with the peptide backbone via CMAP and the χ1 and χ2 dihedral parameters leads to improved treatment of the analyzed nonbond interactions. This highlights the importance of proper treatment of the internal covalent components in modeling nonbond interactions with mol. mechanics FFs.
    54. 54
      Bannwarth, C.; Ehlert, S.; Grimme, S. GFN2-xTB─An Accurate and Broadly Parametrized Self-Consistent Tight-Binding Quantum Chemical Method with Multipole Electrostatics and Density-Dependent Dispersion Contributions. J. Chem. Theory Comput. 2019, 15, 1652– 1671,  DOI: 10.1021/acs.jctc.8b01176
      54
      GFN2-xTB-An Accurate and Broadly Parametrized Self-Consistent Tight-Binding Quantum Chemical Method with Multipole Electrostatics and Density-Dependent Dispersion Contributions
      Bannwarth, Christoph; Ehlert, Sebastian; Grimme, Stefan
      Journal of Chemical Theory and Computation (2019), 15 (3), 1652-1671CODEN: JCTCCE; ISSN:1549-9618. (American Chemical Society)
      An extended semiempirical tight-binding model is presented, which is primarily designed for the fast calcn. of structures and noncovalent interactions energies for mol. systems with roughly 1000 atoms. The essential novelty in this so-called GFN2-xTB method is the inclusion of anisotropic second order d. fluctuation effects via short-range damped interactions of cumulative at. multipole moments. Without noticeable increase in the computational demands, this results in a less empirical and overall more phys. sound method, which does not require any classical halogen or hydrogen bonding corrections and which relies solely on global and element-specific parameters (available up to radon, Z = 86). Moreover, the at. partial charge dependent D4 London dispersion model is incorporated self-consistently, which can be naturally obtained in a tight-binding picture from second order d. fluctuations. Fully anal. and numerically precise gradients (nuclear forces) are implemented. The accuracy of the method is benchmarked for a wide variety of systems and compared with other semiempirical methods. Along with excellent performance for the "target" properties, we also find lower errors for "off-target" properties such as barrier heights and mol. dipole moments. High computational efficiency along with the improved physics compared to it precursor GFN-xTB makes this method well-suited to explore the conformational space of mol. systems. Significant improvements are furthermore obsd. for various benchmark sets, which are prototypical for biomol. systems in aq. soln.
    55. 55
      Melo, M. C.; Bernardi, R. C.; Rudack, T.; Scheurer, M.; Riplinger, C.; Phillips, J. C.; Maia, J. D.; Rocha, G. B.; Ribeiro, J. V.; Stone, J. E. NAMD Goes Quantum: An Integrative Suite for Hybrid Simulations. Nat. Methods 2018, 15, 351– 354,  DOI: 10.1038/nmeth.4638
      55
      NAMD goes quantum: an integrative suite for hybrid simulations
      Melo, Marcelo C. R.; Bernardi, Rafael C.; Rudack, Till; Scheurer, Maximilian; Riplinger, Christoph; Phillips, James C.; Maia, Julio D. C.; Rocha, Gerd B.; Ribeiro, Joao V.; Stone, John E.; Neese, Frank; Schulten, Klaus; Luthey-Schulten, Zaida
      Nature Methods (2018), 15 (5), 351-354CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)
      Hybrid methods that combine quantum mechanics (QM) and mol. mechanics (MM) can be applied to studies of reaction mechanisms in locations ranging from active sites of small enzymes to multiple sites in large bioenergetic complexes. By combining the widely used mol. dynamics and visualization programs NAMD and VMD with the quantum chem. packages ORCA and MOPAC, we created an integrated, comprehensive, customizable, and easy-to-use suite (http://www.ks.uiuc.edu/Research/qmmm). Through the QwikMD interface, setup, execution, visualization, and anal. are streamlined for all levels of expertise.
    56. 56
      Bursch, M.; Neugebauer, H.; Grimme, S. Structure Optimisation of Large Transition-Metal Complexes with Extended Tight-Binding Methods. Angew. Chem., Int. Ed. 2019, 58, 11078– 11087,  DOI: 10.1002/anie.201904021
      56
      Structure Optimisation of Large Transition-Metal Complexes with Extended Tight-Binding Methods
      Bursch, Markus; Neugebauer, Hagen; Grimme, Stefan
      Angewandte Chemie, International Edition (2019), 58 (32), 11078-11087CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Large transition-metal complexes are used in numerous areas of chem. Computer-aided theor. investigations of such complexes are limited by the sheer size of real systems often consisting of hundreds to thousands of atoms. Accordingly, the development and thorough evaluation of fast semiempirical quantum chem. methods that are universally applicable to a large part of the periodic table is indispensable. Herein, we report on the capability of the recently developed GFNn-xTB method family for full quantum-mech. geometry optimization of medium to very large transition-metal complexes and organometallic supramol. structures. The results for a specially compiled benchmark set of 145 diverse closed-shell transition-metal complex structures for all metals up to Hg are presented. Further the GFNn-xTB methods are tested on three established benchmark sets regarding reaction energies and barrier heights of organometallic reactions.
    57. 57
      Maruno, Y.; Shoji, M.; Koizumi, K.; Nishiyama, Y.; Kitagawa, Y.; Kawakami, T.; Okumura, M.; Yamaguchi, K. Theoretical Studies on Magnetic Interactions between Ni (II) Ions in Urease. Polyhedron 2005, 24, 2778– 2783,  DOI: 10.1016/j.poly.2005.03.161
      57
      Theoretical studies on magnetic interactions between Ni(II) ions in urease
      Maruno, Yusuke; Shoji, Mitsuo; Koizumi, Kenichi; Nishiyama, Yusuke; Kitagawa, Yasutaka; Kawakami, Takashi; Okumura, Mitsutaka; Yamaguchi, Kizashi
      Polyhedron (2005), 24 (16-17), 2778-2783CODEN: PLYHDE; ISSN:0277-5387. (Elsevier B.V.)
      The chem. species of bridge oxygen (WB) in active site of urease was examd. in terms of a magnetic interaction between Ni(II) ions. The effective exchange integrals (Jab) values between Ni(II) ions were calcd. by using three candidates of WB, i.e., H2O, OH- and O2-. The unrestricted hybrid d. functional theory (UHDFT) calcns. were carried out using the smallest models and realistic models. In the case of the smallest models, the exptl. Jab value (-6.3 cm-1) was a middle value of WB = H2O (-2.6 cm-1) and WB = OH- (-11.4 cm-1), while we could not obtain an appropriate electronic structure using the O2--bridge model. The Jab became -16.7 cm-1 by using more realistic OH--bridge model, while one spin d. on the Ni2 ion was delocalized to Asp360 in case of H2O-bridge model. Natural orbital analyses revealed that the magnetic orbital through Lys-bridge delocalized to Asp360 and it seemed to assoc. with the spin transfer. From the results, the OH--bridge seemed to be the most likely candidate for the structure of active site in urease.
    58. 58
      Kitagawa, Y.; Saito, T.; Ito, M.; Nakanishi, Y.; Shoji, M.; Koizumi, K.; Yamanaka, S.; Kawakami, T.; Okumura, M.; Yamaguchi, K. Geometry Optimization Method Based on Approximate Spin Projection and its Application to F2, CH2, CH2OO, and Active Site of Urease. Int. J. Quantum Chem. 2007, 107, 3094– 3102,  DOI: 10.1002/qua.21456
      58
      Geometry optimization method based on approximate spin projection and its application to F2, CH2, CH2OO, and active site of urease
      Kitagawa, Yasutaka; Saito, Toru; Ito, Masahide; Nakanishi, Yasuyuki; Shoji, Mitsuo; Koizumi, Kenichi; Yamanaka, Shusuke; Kawakami, Takashi; Okumura, Mitsutaka; Yamaguchi, Kizashi
      International Journal of Quantum Chemistry (2007), 107 (15), 3094-3102CODEN: IJQCB2; ISSN:0020-7608. (John Wiley & Sons, Inc.)
      A new geometry optimization method based on an approx. spin projection (AP) procedure is proposed to eliminate a spin contamination effect in an optimized structure on a low spin (LS) state of a broken symmetry (BS) method. First, an energy gradient with the AP (AP gradient) is derived and it is applied to the geometry optimization of F2, CH2, and CH2OO in order to obtain their structures without the spin contamination. The optimization method corrects H-C-H angle of the CH2 about 10° in comparison with the BS method, and indicates that the spin contamination in optimized geometry of the BS LS states is considerably large. Next, an active site of Urease that consists of two Ni(II) ions is optimized by the AP method. The result indicates that structure of the active site is sensitive to a water mol. called W3.
    59. 59
      Frisch, M. J.; Gaussian 16, Revision B.01; Gaussian Inc.: Wallingford, CT, 2016.
      There is no corresponding record for this reference.
    60. 60
      Bussi, G.; Laio, A.; Parrinello, M. Equilibrium Free Energies from Nonequilibrium Metadynamics. Physi. Rev. Lett. 2006, 96, 090601,  DOI: 10.1103/PhysRevLett.96.090601
      60
      Equilibrium Free Energies from Nonequilibrium Metadynamics
      Bussi, Giovanni; Laio, Alessandro; Parrinello, Michele
      Physical Review Letters (2006), 96 (9), 090601/1-090601/4CODEN: PRLTAO; ISSN:0031-9007. (American Physical Society)
      We propose a new formalism to map history-dependent metadynamics in a Markovian process. We apply this formalism to model Langevin dynamics and det. the equil. distribution of a collection of simulations. We demonstrate that the reconstructed free energy is an unbiased est. of the underlying free energy and anal. derive an expression for the error. The present results can be applied to other history-dependent stochastic processes, such as Wang-Landau sampling.
    61. 61
      Castro, C. B.; Ferreira, M. P.; Caterina, G. M. N. Metalloenzyme Mechanisms Correlated to Their Turnover Number and Metal Lability. Curr. Res. Chem. Biol. 2021, 1, 100004,  DOI: 10.1016/j.crchbi.2021.100004
      There is no corresponding record for this reference.
    62. 62
      Krajewska, B.; van Eldik, R.; Brindell, M. Temperature- and Pressure-Dependent Stopped-Flow Kinetic Studies of Jack Bean Urease. Implications for the Catalytic Mechanism. J. Biol. Inorg. Chem. 2012, 17, 1123– 1134,  DOI: 10.1007/s00775-012-0926-8
      62
      Temperature- and pressure-dependent stopped-flow kinetic studies of jack bean urease. Implications for the catalytic mechanism
      Krajewska, Barbara; van Eldik, Rudi; Brindell, Malgorzata
      JBIC, Journal of Biological Inorganic Chemistry (2012), 17 (7), 1123-1134CODEN: JJBCFA; ISSN:0949-8257. (Springer)
      Urease, a Ni-contg. metalloenzyme, features an activity that has profound medical and agricultural implications. The mechanism of this activity, however, has not been as yet thoroughly established. Accordingly, to improve its understanding, in this study we analyzed the steady-state kinetic parameters of the enzyme (jack bean), Km and kcat, measured at different temps. and pressures. Such an anal. is useful as it provides information on the mol. nature of the intermediate and transition states of the catalytic reaction. We measured the parameters in a noninteracting buffer using a stopped-flow technique at 15-35° and in the pressure range 5-132 MPa, the pressure-dependent measurements being the first of their kind performed for urease. While temp. enhanced the activity of urease, pressure inhibited the enzyme; the inhibition was biphasic. Analyzing Km provided the characteristics of the formation of the ES complex, and analyzing kcat, the characteristics of the activation of ES. From the temp.-dependent measurements, the energetic parameters were derived, i.e. thermodn. ΔHo and ΔSo for ES formation, and kinetic ΔH≠ and ΔS≠ for ES activation, while from the pressure-dependent measurements, the binding ΔVb and activation vols. were detd. The thermodn. and activation parameters obtained are discussed in terms of the current proposals for the mechanism of the urease reaction, and they are found to support the mechanism proposed by Benini et al., in which the Ni-Ni bridging hydroxide-not the terminal hydroxide-is the nucleophile in the catalytic reaction.
    63. 63
      El-Hefnawy, M. E.; Sakran, M.; Ismail, A. I.; Aboelfetoh, E. F. Extraction, Purification, Kinetic and Thermodynamic Properties of Urease from Germinating Pisum Sativum L. seeds. BMC Biochem. 2014, 15, 15,  DOI: 10.1186/1471-2091-15-15
      63
      Extraction, purification, kinetic and thermodynamic properties of urease from germinating Pisum sativum L. seeds
      El-Hefnawy, Mohamed E.; Sakran, Mohamed; Ismail, Ali I.; Aboelfetoh, Eman Fahmy
      BMC Biochemistry (2014), 15 (), 15/1-15/8, 8 pp.CODEN: BBMIB3 ISSN:. (BioMed Central Ltd.)
      Background: Urease, one of the highly efficient known enzymes, catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The present study aimed to ext. urease from pea seeds (Pisum sativum L). The enzyme was then purified in three consequence steps: acetone pptn., DEAE-cellulose ion-exchange chromatog. and gel filtration chromatog. (Sephacryl S-200 column). Results: The purifn. fold was 12.85 with a yield of 40%. The mol. wt. of the isolated urease was estd. by chromatog. to be 269,000 Daltons. Maximum urease activity (190 U/g) was achieved at the optimum conditions of 40°C and pH of 7.5 after 5 min of incubation. The kinetic parameters, Km and Vmax, were estd. by Lineweaver-Burk fits and found to be 500 mM and 333.3 U/g, resp. The thermodn. consts. of activation, ΔH, Ea, and ΔS, were detd. using Arrhenius plot and found to be 21.20 kJ/mol, 23.7 kJ/mol and 1.18 kJ/mol/K, resp. Conclusions: Urease was purified from germinating Pisum sativum L. seeds. The purifn. fold, yield and mol. wt. were detd. The effects of pH, concn. of enzyme, temp., concn. of substrate and storage period on urease activity were examd. This may provide an insight on the various aspects of the property of the enzyme. The significance of extg. urease from different sources could play a good role in understanding the metab. of urea in plants.
    64. 64
      Musiani, F.; Arnofi, E.; Casadio, R.; Ciurli, S. Structure-Based Computational Study of the Catalytic and Inhibition Mechanisms of Urease. J. Biol. Inorg. Chem. 2001, 6, 300– 314,  DOI: 10.1007/s007750000204
      64
      Structure-based computational study of the catalytic and inhibition mechanisms of urease
      Musiani, Francesco; Arnofi, Elisa; Casadio, Rita; Ciurli, Stefano
      JBIC, Journal of Biological Inorganic Chemistry (2001), 6 (3), 300-314CODEN: JJBCFA; ISSN:0949-8257. (Springer-Verlag)
      The viability of different mechanisms of catalysis and inhibition of the nickel-contg. enzyme urease was explored using the available high-resoln. structures of the enzyme isolated from Bacillus pasteurii in the native form and inhibited with several substrates. The structures and charge distribution of urea, its catalytic transition state, and three enzyme inhibitors were calcd. using ab initio and d. functional theory methods. The DOCK program suite was employed to det. families of structures of urease complexes characterized by docking energy scores indicative of their relative stability according to steric and electrostatic criteria. Adjustment of the parameters used by DOCK, in order to account for the presence of the metal ion in the active site, resulted in the calcn. of best energy structures for the nickel-bound inhibitors β-mercaptoethanol, acetohydroxamic acid, and diamidophosphoric acid. These calcd. structures are in good agreement with the exptl. detd. structures, and provide hints on the reactivity and mobility of the inhibitors in the active site. The same docking protocol was applied to the substrate urea and its catalytic transition state, in order to shed light onto the possible catalytic steps occurring at the binuclear nickel active site. These calcns. suggest that the most viable pathway for urea hydrolysis involve a nucleophilic attack by the bridging, and not the terminal, nickel-bound hydroxide onto a urea mol., with active site residues playing important roles in orienting and activating the substrate, and stabilizing the catalytic transition state.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.1c10200.

    • Figure S1, active site models for 1 and 2 excluding H222, H323, and A366 with key distances and spin density; Cartesian coordinates of these models obtained with the UB3LYP/def2-SVP level; the created topology and parameter files; Figure S2, RMSDs with respect to the initial conformations during a 10 ns classical MD simulation for 1 and 2; Figure S3, overlay of smaller models (1, 2, and 3) optimized in the singlet and quintet states at GFN2-xTB with respect to the structure obtained with the broken-symmetry DFT method; Figure S4, structure and Cartesian coordinates of the QM region for 1 optimized in the open-shell singlet state at the UB3LYP/def2-SV(P) level; Figure S5, potential energy profile of the dissociation of W2; Cartesian coordinates for all stationary points of the QM-only cluster calculations optimized at the GFN2-xTB and UB3LYP levels; Tables S1–S5, key bond distance for all stationary points obtained from QM-only cluster and QM/MM metadynamics calculations; full citation for ref (59) (PDF)


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