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Spectroscopic and Thermodynamic Characterization of the E151D and E151A Altered Leucine Aminopeptidases from Aeromonas proteolytica^†

Krzysztof P. Bzymek,^¶ Sabina I. Swierczek,^¶ Brian Bennett,*^§ and Richard C. Holz*^¶

Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300

Inorg. Chem., 2005, 44 (23), pp 8574–8580

DOI: 10.1021/ic051034g

Publication Date (Web): September 24, 2005

†

Abbreviations: rAAP, recombinant leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus); BuBA, 1-butaneboronic acid; CPG₂, carboxypeptidase G₂; GCP II, glutamate carboxypeptidase II; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IPTG, isopropyl-β-D-thiogalactopyranoside; ICP-AES, inductively coupled plasma-atomic emission spectrometry; ITC, Isothermal Titration Calorimetry; L-pNA, l-leucine-p-nitroanilide; Tricine, N-tris[hydroxymethyl]methylglycine; WT, wild type.

Utah State University.

To whom correspondence should be addressed. Phone: (435) 797-2609. Fax: (435) 797-3390. E-mail: rholz@cc.usu.edu.

University of Wisconsin, Milwaukee.

Abstract

Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 Å in carboxypeptidase G₂ and 3.30 and 3.63 Å in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by UV−vis and electron paramagnetic resonance (EPR) spectroscopy. UV−vis spectroscopy of Co(II)-substituted E151A-AAP did not reveal any significant changes in the electronic absorption spectra. However UV−vis spectra of mono- and dicobalt(II) E151D-AAP exhibited a lower molecular absorptivity compared to AAP (23 and 43 M^-¹ cm^-¹ vs. 56 and 109 M^-¹ cm^-¹ for E151D-AAP and AAP, respectively) suggesting both Co(II) ions reside in distorted octahedral coordination geometry in E151D-AAP. EPR spectra of [Co_(E151D-AAP)], [ZnCo(E151D-AAP)], and [(CoCo(E151D-AAP)] were identical, with g = 2.35, g = 2.19, and E/D = 0.19, similar to [CoCo(AAP)]. On the other hand, the EPR spectrum of [Co_(E151A-AAP)] was best simulated assuming the presence of two species with (i) g_x_,_y = 2.509, g_z = 2.19, E/D = 0.19, A = 0.0069 cm^-¹ and (ii) g_x_,_y = 2.565, g_z = 2.19, E/D = 0.20, A = 0.0082 cm^-¹ indicative of a five- or six-coordinate species. Isothermal titration calorimetry experiments revealed a large decrease in Zn(II) affinities, with K_d values elevated by factors of 850 and 24 000 for the first metal binding events of E151D- and E151A-AAP, respectively. The combination of these data indicates that E151 serves to stabilize the dinuclear active site of AAP.