Related Experiment Video
Updated: Mar 26, 2026

RhoC GTPase Activation Assay
Published on: August 22, 2010
(19)F NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA-Catalyzed GTP
Yi Jin1, Robert W Molt2, Jonathan P Waltho3,4
1Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK.
Abstract:
Molecular details for RhoA/GAP catalysis of the hydrolysis of GTP to GDP are poorly understood. We use (19)F 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.
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