¹⁹F 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.

The effect of magnesium ions on triphosphate hydrolysis

“…The mechanisms of these reactions and the importance of the different factors affecting the fundamental mechanistic preferences (such as environment or leaving/spectator group effects) have been hotly debated [6,7], without reaching a mechanistic consensus. For example, in the case of GTP hydrolysis by GTPases such as Ras GTPase, arguments have been put forward in favor of phosphorane intermediates and concerted reaction pathways [8], of substrate-assisted catalysis [9][10][11][12][13], of general base catalysis with the involvement of active site residues [14][15][16][17], or the possibility that no deprotonation of the nucleophile is required to drive the reaction [6,18]. A similar lack of mechanistic clarity exists for many other enzymes that catalyze phosphoryl transfer reactions (see discussion in e.g.…”

Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes

“…Reduction in the number of H-bond partners generally results in upfield shift of 19 F resonances, as shown clearly in a comparison of the G6P and the 2-deoxy G6P Fig. 14 a 1D 19 F NMR spectra of: a RhoA/GAPÁGDPÁAlF 4 -TSA complex with four fluorines rotationally averaged [44,48]. b RhoA/GAPÁGDPÁMgF 3 -TSA complex with three fluorines resolved [44].…”

“…They use an aspartyl phosphate intermediate, whose TS for hydrolysis is mimicked by an octahedral AlF 4 -. An axial water oxygen forms short H-bonds to an invariant glutamate (2.5 ± 0.1 Å ) and to a threonine carbonyl (2.57 ± 0.05 Å ), which clearly orientate and polarize the water for ''in-line'' attack on the aspartyl phosphate [44]. …”

“…14a, b) [44,50,51,59,60]. In the context of TSAs and GSAs, the chemical shifts of 19 F resonances provide a key measure of interactions between MF x moieties and their protein hosts, and report the electronic environment of the fluorine nuclei.…”

“…In the context of TSAs and GSAs, the chemical shifts of 19 F resonances provide a key measure of interactions between MF x moieties and their protein hosts, and report the electronic environment of the fluorine nuclei. When combined with NMR computations, they also act as indirect reporters of changes in electronic environment experienced by phosphoryl oxygen atoms in transfer reaction TSs [44,61,62]. 19 F NMR resonances display a high degree of dispersion and are calculable with good precision from QM analysis of electronic distribution [37,51,63], showing resonances strongly affected by neighboring H-bond donors.…”