Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8,000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, molecular-dynamics simulations and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events: phosphoryl transfer (>105-fold) and lid opening (103-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a preorganized active site.
Bibliographical noteFunding Information:
We dedicate this manuscript to Tom Alber, a truly amazing and inspiring scientist, and a close friend who will live in our hearts forever; his creativity, joy and generosity have deeply influenced several generations of scientists. We are grateful to the staff at the Advanced Light Source–Berkeley Center for Structural Biology and the Advanced Photon Source (APS) for support, Y. Xiong, T. Lang, P. Afonine, R. Read and mentors from the Collaborative Computational Project No. 4 School at APS (2011) for advice in the refinement of X-ray data, members of the C. Miller laboratory for handling crystals, the staff at the National Energy Research Scientific Computing Center, and K.A. Johnson for assistance with the KinTek Explorer software and fitting kinetic data. We thank P. Varilly (University of Cambridge) for kindly providing scripts. This work was supported by the Howard Hughes Medical Institute (HHMI), the Office of Basic Energy Sciences, Catalysis Science Program, US Department of Energy (award DE-FG02-05ER15699), the US National Institutes of Health (RO1-GM100966) and the Teragrid (XSEDE) allocation TG-MCB090166 (D.K.). R.O. is supported as an HHMI Fellow of the Damon Runyon Cancer Research Foundation (DRG-2114-12).
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