Characterization of Enzyme Motions by Solution NMR Relaxation Dispersion

• Published in: Accounts of chemical research 2008-02, Vol.41 (2), p.214-221
• Main Authors: Loria, J. Patrick, Berlow, Rebecca B, Watt, Eric D
• Format: Article
• Language: English
• Subjects: Enzymes - chemistry; Enzymes - metabolism; Hydrogen Bonding; Ligands; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Thermodynamics
• ISSN: 0001-4842; 1520-4898
• DOI: 10.1021/ar700132n

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 conformatio