GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the Gαi subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4 – and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) “undruggable” GTPases like Ras.

The subset of catalytically competent conformations can be significantly small in comparison with the full conformational landscape of enzyme–substrate complexes. In some enzymes, the probability of finding a reactive conformation can account for up to 4 kcal/mol of activation barrier, even when the substrate remains tightly bound. In this study, we sampled conformations of human pancreatic α-amylase with bound substrate in a molecular dynamics (MD) simulation of over 100 ns and calculated energy profiles along the reaction coordinate. We found that reactive states require a hydrogen bond between a buried water molecule and E233, which is the general acid in the glycolysis mechanism. The effect of this single, nonreactive, intermolecular interaction is as important as the correct positioning and orientation of the reacting residues to achieve a competent energy barrier. This hydrogen bond increases the acidity of E233, facilitating proton transfer to the glycosidic oxygen. In the MD simulation, this required hydrogen bond was observed in more than half of the microstates, indicating that human pancreatic α-amylase is efficient at maintaining this important interaction in the reactant state. Furthermore, this hydrogen bond formed and vanished on a subnanosecond time scale. Interactions between the reacting groups also change on this time scale. All of these changes led to instantaneous activation energy oscillations from 9.3 to 28.3 kcal/mol on a much smaller time scale in comparison to the turnover rate. These results are in agreement with the observed kinetics being determined by a few transient conformations that require low energy barriers.

One enzyme, one substrate, but two different reaction mechanisms: HIV-1 protease follows different reaction mechanisms depending on its instantaneous conformation.