(FNMR)-F-19 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.