Characterization of Enzyme Motions by Solution NMR Relaxation Dispersion

Patrick Loria received his B.S. degree in Chemistry from The George Washington University in Washington, D.C. and a Ph.D. in Biochemistry working with Tom Nowak at the University of Notre Dame. He was a NIH postdoctoral fellow with Professor Arthur Palmer at Columbia University until 2000. He joined the Chemistry faculty at Yale University in 2001. His research efforts focus on solution NMR and biochemistry for characterization of enzymes and proteins involved in bacterial pathogenesis.

Rebecca Berlow received her B.A. in Chemistry from the Johns Hopkins University in 2005. She is currently pursuing her Ph.D. in Molecular Biophysics and Biochemistry at Yale University in the laboratory of Patrick Loria. At present, her research focuses on characterizing allosteric mechanisms in prokaryotic response regulator proteins involved in antibiotic resistance, in addition to continuing studies on the role of motional processes in enzyme function.

Eric Watt received his B.S. in Chemistry from the University of Michigan in 2005. He is currently pursuing his Ph.D. in Chemistry at Yale University in the Loria laboratory. His current research focuses on utilizing NMR relaxation experiments to characterize protein dynamics and folding.

In many enzymes, conformational changes that occur along the reaction coordinate can pose a bottleneck to the rate of conversion of substrates to products. Characterization of these rate-limiting protein motions is essential for obtaining a full understanding of enzyme-catalyzed reactions. Solution NMR experiments such as the Carr−Purcell−Meiboom−Gill (CPMG) spin-echo or off-resonance R_1ρ pulse sequences enable quantitation of protein motions in the time range of microseconds to milliseconds. These experiments allow characterization of the conformational exchange rate constant, k_ex, the equilibrium populations of the relevant conformations, and the chemical shift differences (Δω) between the conformations.

The CPMG experiments were applied to the backbone N−H positions of ribonuclease A (RNase A). To probe the role of dynamic processes in the catalytic cycle of RNase A, stable mimics of the apo enzyme (E), enzyme−substrate (ES) complex, and enzyme−product (EP) complex were formed. The results indicate that the ligand has relatively little influence on the kinetics of motion, which occurs at 1700 s^–1 and is the same as both k_cat, and the product dissociation rate constant. Instead, the effect of ligand is to stabilize one of the pre-existing conformations. Thus, these NMR experiments indicate that the conformational change in RNase A is ligand-stabilized and does not appear to be ligand-induced. Further evidence for the coupling of motion and enzyme function comes from the similar solvent deuterium kinetic isotope effect on k_ex derived from the NMR measurements and k_cat from enzyme kinetic studies. This isotope effect of ~2 depends linearly on solvent deuterium content suggesting the involvement of a single proton in RNase A motion and function. Moreover, mutation of His48 to alanine eliminates motion in RNase A and decreases the catalytic turnover rate indicating the involvement of His48, which is far from the active site, in coupling motion and function.

For the enzyme triosephosphate isomerase (TIM), the opening and closing motion of a highly conserved active site loop (loop 6) has been implicated in many studies to play an important role in the catalytic cycle of the enzyme. Off-resonance R_1ρ experiments were performed on TIM, and results were obtained for amino acid residues in the N-terminal (Val167), and C-terminal (Lys174, Thr177) portions of loop 6. The results indicate that all three loop residues move between the open and closed conformation at about 10000 s^–1, which is the same as the catalytic rate constant. The O^η atom of Tyr208 provides a hydrogen bond to stabilize the closed form of loop 6 by interacting with the amide nitrogen of Ala176; these atoms are outside of hydrogen bonding distance in the open form of the enzyme. Mutation of Tyr208 to phenylalanine results in significant loss of catalytic activity but does not appear to alter the kex value of the N-terminal part of loop 6. Instead, removal of this hydrogen bond appears to result in an increase in the equilibrium population of the open conformer of loop 6, thereby resulting in a loss of activity through a shift in the conformational equilibrium of loop 6.

Solution NMR relaxation dispersion experiments are powerful experimental tools that can elucidate protein motions with atomic resolution and can provide insight into the role of these motions in biological function.