19F NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA-Catalyzed GTP Hydrolysis

Molecular details for RhoA/GAP catalysis of the hydrolysis of GTP to GDP are poorly understood. We use 19F NMR chemical shifts in the MgF3− transition state analogue (TSA) complex as a spectroscopic reporter to indicate electron distribution for the γ‐PO3− oxygens in the corresponding TS, implying that oxygen coordinated to Mg has the greatest electron density. This was validated by QM calculations giving a picture of the electronic properties of the transition state (TS) for nucleophilic attack of water on the γ‐PO3− group based on the structure of a RhoA/GAP‐GDP‐MgF3− TSA complex. The TS model displays a network of 20 hydrogen bonds, including the GAP Arg85′ side chain, but neither phosphate torsional strain nor general base catalysis is evident. The nucleophilic water occupies a reactive location different from that in multiple ground state complexes, arising from reorientation of the Gln‐63 carboxamide by Arg85′ to preclude direct hydrogen bonding from water to the target γ‐PO3− group. Guanosine 5′‐triphosphate hydrolysis by the small G protein, RhoA, was analyzed by combining X‐ray, 19F NMR, and DFT calculations. A transition‐state model based on 19F NMR spectra is proposed for nucleophilic attack of water on GTP, founded on an extensive network of 20 hydrogen bonds. This network disrupts inhibitory hydrogen bonds that stabilize an unreactive ground state conformation.