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

Abstract

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.