Abstract
Protein conformational changes are often essential for enzyme catalysis and, in several cases, are shown to be the limiting factor for overall catalytic speed. However, a structural understanding of the corresponding transition states, needed to rationalize the kinetics, remains obscure due to their transient nature. Here, we determine the transition state ensemble of the rate-limiting conformational transition in the enzyme adenylate kinase through a synergistic approach between experimental high-pressure NMR relaxation during catalysis and molecular dynamics simulations. By comparing homologous kinases that evolved under ambient or high pressure in the deep sea, we detail transition state ensembles that differ in solvation as directly measured by the pressure dependence of catalysis. Capturing transition state ensembles begins to complete the catalytic energy landscape that is generally characterized by the structures of all intermediates and the frequencies of transitions among them.