Enzymatic evolution driven by entropy production

Arango-Restrepo, Andrés; Rubı́, J. M.

doi:10.1101/319814

Cited by 1 publication

(1 citation statement)

References 27 publications

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“…The presence of the GAP heavily restricts the conformational space of this residue, leading to the side chain to sample a single conformation that interacts directly with the nucleophilic water molecule at the Michaelis complex and transition states for the phosphoryl transfer step (note that as shown in Figures and S17, the conformational space of both the water molecule and the Gln side chain is greatly reduced at the Michaelis complexes for Ras both with and without GAP). As a consequence, the lower entropy of the Michaelis complex in the case of RasGAP reduces the activation barrier compared to the intrinsic reaction (for a broader discussion of the issue of conformational sampling and entropy in enzyme catalysis, see, e.g., refs and − among others). The average RMSD of this side chain at the Michaelis complex is 1.26 ± 0.08 and 0.28 ± 0.02 Å in the case of Ras and RasGAP, respectively.…”

Section: Results and Discussionmentioning

confidence: 99%

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases

et al. 2019

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GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the Gαi subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4 – and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) “undruggable” GTPases like Ras.

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