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 19 FNMR chemical shifts in the MgF 3 À transition state analogue (TSA) complex as aspectroscopic reporter to indicate electron distribution for the g-PO 3À oxygens in the corresponding TS, implying that oxygen coordinated to Mg has the greatest electron density.This was validated by QM calculations giving apicture of the electronic properties of the transition state (TS) for nucleophilic attack of water on the g-PO 3À group based on the structure of aRhoA/GAP-GDP-MgF 3 À TSA complex. The TS model displays anetwork 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 ar eactive location different from that in multiple ground state complexes,a rising from reorientation of the Gln-63 carboxamide by Arg85' to preclude direct hydrogen bonding from water to the target g-PO 3À group.Hydrolysis of guanosine triphosphate (GTP) by small G proteins (GTPases) of the Ras oncogene superfamily initiates ac onformational change that results in On/Off signaling for aw ide range of cellular activities.[1] Slow spontaneous GTP hydrolysis in water (10 À9 s À1 )isaccelerated by up to 5orders of magnitude by GTPases,while cooperative GTPase activating proteins (GAPs) further accelerate hydrolysis by 3t o5 orders of magnitude,raising k cat to 5-30 s À1

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

“…[26a] More recent work has sought to establish the extent to which MgF3 -resembles PTx in the TS for GTP hydrolysis catalyzed by the RhoA/RhoGAP complex. [20] Specifically, this study, which employed DFT calculations on a very large active site model, containing 91 heavy atoms, demonstrated that the observed 19 F chemical shifts for the RhoA·RhoGAP·GDP·MgF3 -complex can indeed be interpreted as indirect measures of the relative electron densities of the cognate oxygen atoms in the "true" TS for attack of water on the terminal phosphate of GTP. [20] 6.4 AlF3 complexes.…”

“…[43] More recently, computational methods have also validated the idea that tbp MFx structures are analogs of the phosphoryl group in the "true" transition states for enzyme-catalyzed reactions, and provide useful information on the extent to which MFx moieties resemble ground states or TSs in enzyme-catalyzed PTx. [20] Although the covalent character of P-O and M-F bonds is very different in the GS and (most likely) the TS, these calculations demonstrate that differences in NMR 19 F chemical shifts do provide insights into the environments experienced by the oxygen atoms in the "true" TS for the reaction.…”

“…[46] Larger REVIEW 11 models can also be built that include "second shell" residues, which form H-bonds to the initial set of inner residues. [20] In an alternative approach, which avoids the need to place artificial coordinate restraints on atoms in the QM region, the complete system is modeled using QM/MM methodologies. [47] Here the QM region is embedded in the rest of the protein and solvent, with the additional atoms (in an MM region) being described by classical potential energy functions that depend on "force field" parameters.…”

“…These residues clearly orientate and polarize the water for "in-line" attack on the aspartyl phosphate (Section 8.3). [20] …”

“…When combined with calculations (Section 6.3), they can also act as indirect reporters of the changes in electronic environment experienced by phosphoryl oxygen atoms at the TS for the transfer reaction. [20,36] 19 F resonances display a high degree of dispersion and are predictable with good precision from quantum calculations of electronic distribution. [37] The average chemical shifts of resonances from AlFx, MgFx and BeFx species in aqueous solution differ (-154, -156, and -169 ppm, respectively), but a wide spread of individual shifts is observed in complexes with proteins.…”