Abstract
Catalytic function of many enzymes is comprised of a number of microscopic steps including enzyme structural rearrangements and the chemical steps, in which bonds of the substrate(s) are broken/made to synthesize the product(s). Separation of these processes is a challenging task that hinders our understanding of enzyme catalysis. We have used nuclear magnetic resonance (NMR) and rapid-quenching techniques to independently study these processes in adenylate kinase (ADK) and to characterize their energetic contribution at atomic resolution. Adenylate kinase is an important enzyme that catalyzes a reversible reaction: 2ADP ↔ ATP +AMP, and is involved in maintaining energy homeostasis in a cell. Two binding sites of ADK and three different ligands result in a variety of biochemical states (determined by the bound ligands). Each of these states has different dynamic properties making the overall structural dynamics of ADK complex. We have tackled this problem by engineering a loss-of-function ADK mutant (with greatly reduced rate of turnover), which allowed us to “trap” the enzyme in well-defined biochemical states. Our results lead to a detailed energy profile of ADK, providing insights into the molecular mechanism of its functioning. This work reveals fundamental principles of enzyme catalysis and highlights the role of protein's intrinsic dynamics.