title: (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 (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.
authors.affiliation: Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK.
authors.forename: Yi
authors.identifier: http://orcid.org/0000-0002-6927-4371
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN, 46202, USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Molt
authors.affiliation: Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK. j.waltho@manchester.ac.uk.|Manchester Institute of Biotechnology, Manchester, M1 7DN, UK. j.waltho@manchester.ac.uk.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN, 46202, USA. RichardsN14@cardiff.ac.uk.|School of Chemistry, Cardiff University, Cardiff, CF10 3AT, UK. RichardsN14@cardiff.ac.uk.
authors.forename: Nigel G J
authors.initials: NG
authors.lastname: Richards
authors.affiliation: Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK. g.m.blackburn@sheffield.ac.uk.
authors.forename: G Michael
authors.initials: GM
authors.lastname: Blackburn
mesh_terms: D005461:Fluorine / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D005461:Fluorine; D006160:Guanosine Triphosphate; D020742:rhoA GTP-Binding Protein
keywords: 19F NMR; GTP hydrolases; enzyme catalysis; phosphoryl transfer; reaction mechanisms
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