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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 Mg(2+) cofactor activates two distinct molecular events: phosphoryl transfer (>10(5)-fold) and lid opening (10(3)-fold). In contrast, mutation of an essential active site arginine decelerates phosphoryl transfer 10(3)-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.

Protein kinases are obvious drug targets against cancer, owing to their central role in cellular regulation. Since the discovery of Gleevec, a potent and specific inhibitor of Abl kinase, as a highly successful cancer therapeutic, the ability of this drug to distinguish between Abl and other tyrosine kinases such as Src has been intensely investigated but without much success. Using NMR and fast kinetics, we establish a new model that solves this longstanding question of how the two tyrosine kinases adopt almost identical structures when bound to Gleevec but have vastly different affinities. We show that, in contrast to all other proposed models, the origin of Abl's high affinity lies predominantly in a conformational change after binding. An energy landscape providing tight affinity via an induced fit and binding plasticity via a conformational-selection mechanism is likely to be general for many inhibitors.