GTPase are regulatory proteins that are involved in almost all vital cellular processes. Their mechanism and regulation have been the subject of a substantial amount of research effort, both computational and experimental, however the precise mechanism by which GTPases hydrolyze GTP is still unclear. The absence of an obvious general base in the active site to deprotonate the nucleophilic water molecule means the catalytic mechanism utilized by this class of enzymes is still controversial. Here we performed empirical valence bond (EVB) simulations of GTP hydrolysis, comparing both solvent and substrate assisted mechanisms catalyzed by different GTPases: Ras, Rab and the Gαi subunit of a heterotrimeric G-protein, all in the presence and the absence of their GTPases activating proteins (GAP). Our results demonstrate that a general base is not needed in the active site of these enzymes since the preferred mechanism for GTP hydrolysis by these GTPases is a conserved solvent-assisted pathway, in which a proton transfer occurs after the rate-limiting nucleophilic attack of a water molecule at the GTP γ-phosphate. This first step leads to the formation of a short-lived intermediate that tautomerizes to form H2PO4- and GDP as the final products. The obtained free energy values for the solvent assisted mechanism are in agreement with previous experimental results. In conclusion, this work gives detailed mechanistic insights on a catalytic mechanism that has been the subject of decades-old controversy, how Ras and related GTPases hydrolyze GTP. These fundamental biochemical findings can be a new starting point for drug discovery efforts on the (so far) “undruggable” GTPases.

Unraveling the catalytic mechanism of GTP hydrolysis by GTPases using computational approaches

ACS Spring Meeting 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.