Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme

Abstract

Molecular mechanisms of hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus-oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the reaction Ras·GAP·GTP·H 2 O → Ras·GAP·GDP·Pi the highest barriers correspond to the process of regeneration of the active site, but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reactions rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place. Our theoretical predictions are in excellent agreement with available experimental observations on GTP hydrolysis rates.3

The GTPase hGBP1 converts GTP to GMP in two steps via proton shuttle mechanisms

“…The indirect SAC mechanism of GTP hydrolysis in hGBP1 as obtained from our study is somewhat similar to the indirect SAC mechanism proposed for the extensively studied Ras GTPases, in which however only Gln61 is the responsible mediator of catalytic proton transfer from Nu to GTP, 11,12,43,44 whereas recent studies on EF-Tu clearly supported instead a direct SAC mechanism in that case. 10,30 We believe that these mechanistic differences stem from distinctly different active site architectures of the three enzymes.…”

“…Among various GTPases, the catalytic mechanisms of Ras and EF-Tu have been studied extensively. Recent work on these enzymes supports a substrate-assisted catalysis (SAC) pathway, 10–12 in which the substrate catalyzes its own hydrolysis, either directly or indirectly, via a proton shuttle. The GTP hydrolysis is supposed to follow one of three possible paths depending on the bonding patterns of attacking nucleophile (Nu) and leaving group (LG) 1,3 (see Fig.…”

19F NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA‐Catalyzed GTP Hydrolysis

“…The optimized geometry of the TS was obtained using the M06‐2X functional and standard TS search methods (Supporting Information) 12. Importantly, this TS model (Figure 3 c) contained 91 heavy atoms (181 total atoms; Figure 3 b,c), a much larger number than in previous studies (17 atoms;8f 33–37 atoms;10 32 atoms;14 39 atoms8a). The inclusion of the loop atoms for Gly12–Cys16 was essential to obtain a stably protonated state for the sidechain ɛ ‐NH 3 + group of Lys18, and inclusion of the sidechain of Asp59 achieved the correct orientation of the γ‐PO 3 − with excellent superposition of the computed structure on 1ow3 (Figure 3; Supporting Information, Figure S3).…”

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.…”