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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.

Cancer is a serious illness and the second leading cause of death in the US. It is caused by misregulation of the signaling cascades and uncontrolled cell proliferation. Protein kinases are the key players in the regulatory machinery, and in the last 20 years they have become an increasingly important drug targets. The main requirement for such drugs is selectivity: ideally, only one kinase should be affected by an individual drug. The prominent success story is an FDA-approved chronic myeloid leukemia therapeutic Gleevec. Gleevec is a highly specific inhibitor of the Abl kinase and has very little potency towards a closely related Src subfamily of kinases. This specificity is particularly intriguing considering that the drug-binding pockets look nearly identical in these kinases. We combined NMR and pre-steady-state kinetic experiments to monitor Gleevec binding in real time and with residue-specific precision. Our data establish a novel model that fully and quantitatively explains the observed 3000-fold difference in Gleevec's affinities to Abl and Src, solving a long-standing paradox in the field. These results reveal the general principles of kinase-drug interactions and highlight the molecular mechanisms behind nanomolar affinities found in clinically relevant therapeutics.