How Does GAP Catalyze the GTPase Reaction of Ras?:  A Computer Simulation Study

Abstract

The formation of a complex between p21(ras) and GAP accelerates the GTPase reaction of p21(ras) and terminates the signal for cell proliferation. The understanding of this rate acceleration is important for the elucidation of the role of Ras mutants in tumor formation. In principle there are two main options for the origin of the effect of GAP. One is a direct electrostatic interaction between the residues of GAP and the transition state of the Ras-GAP complex and the other is a GAP-induced shift of the structure of Ras to a configuration that increases the stabilization of the transition state. This work examines the relative importance of these options by computer simulations of the catalytic effect of Ras. The simulations use the empirical valence bond (EVB) method to study the GTPase reaction along the alternative associative and dissociative paths. This approach reproduces the trend in the overall experimentally observed catalytic effect of GAP: the calculated effect is 7 +/- 3 kcal/mol as compared to the observed effect of approximately 6.6 kcal/mol. Furthermore, the calculated effect of mutating Arg789 to a nonpolar residue is 3-4 kcal/mol as compared to the observed effect of 4.5 kcal/mol for the Arg789Ala mutation. It is concluded, in agreement with previous proposals, that the effect of Arg789 is associated with its direct interaction with the transition state charge distribution. However, calculations that use the coordinates of Ras from the Ras-GAP complex (referred to here as Ras') reproduce a significant catalytic effect relative to the Ras coordinates. This indicates that part of the effect of GAP involves a stabilization of a catalytic configuration of Ras. This configuration increases the positive electrostatic potential on the beta-phosphate (relative to the corresponding situation in the free Ras). In other words, GAP stabilizes the GDP bound configuration of Ras relative to that of the GTP-bound conformation. The elusive oncogenic effect of mutating Gln61 is also explored. The calculated effect of such mutations in the Ras-GAP complex are found to be small, while the observed effect is very large (8.7 kcal/mol). Since the Ras is locked in its Ras-GAP configuration in our simulations, we conclude that the oncogenic effect of mutation of Gln61 is indirect and is associated most probably with the structural changes of Ras upon forming the Ras-GAP complex. In view of these and the results for the Ras' we conclude that GAP activates Ras by both direct electrostatic stabilization of the transition state and an indirect allosteric effect that stabilizes the GDP-bound form. The present study also explored the feasibility of the associative and dissociative mechanism in the GTPase reaction of Ras. It is concluded that the reaction is most likely to involve an associative mechanism.

Assessing the Influence of Mutation on GTPase Transition States by Using X‐ray Crystallography, 19F NMR, and DFT Approaches

“…Our work therefore supports previous QM/MM studies of the cognate mutation in the Ras/RasGAP complex 8f. Thus, RhoGAP binding delivers additional catalysis through displacement of Tyr34 by the arginine finger, thereby enabling the guanidinium group to diminish electron density on P G by the formation of a stronger H‐bond to O 3G .…”

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

“…The rate acceleration provided by GAPs has been broadly attributed to the insertion of an arginine finger into the active site,17 where it provides electrostatic catalysis,7c, 8f makes the β‐phosphate a better leaving group by hydrogen bonding to the β,γ‐bridging oxygen (O3β), and excludes solvent 17, 18. Our results identify two additional roles for Arg85′.…”

“…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).…”

Why nature really chose phosphate

“…In fact, the realization that Gln cannot be the base led us to examine alternative mechanistic options, where the GTP accepts the proton in either a stepwise or concerted fashion (relative to the actual phosphoryl transfer), and is then hydrolyzed in either an associative or dissociative pathway (see e.g. Glennon et al 2000). The relevant mechanistic considerations ranged from an examination of LFER (Schweins et al 1996a) to considerations of the effect of the arginine finger on the associative and dissociative mechanisms (Glennon et al 2000).…”

“…Glennon et al 2000). The relevant mechanistic considerations ranged from an examination of LFER (Schweins et al 1996a) to considerations of the effect of the arginine finger on the associative and dissociative mechanisms (Glennon et al 2000). In fact, the realization that the central mechanistic questions can only be resolved by theoretical studies (which must of course reproduce the relevant experimental observables) formed the basis for most of our mechanistic studies.…”