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GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.ORCID iD: 0000-0002-6904-2511
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.ORCID iD: 0000-0002-7705-5371
Ruhr Univ Bochum, Dept Biophys, D-44801 Bochum, Germany.
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2019 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 141, no 27, p. 10684-10701Article in journal (Refereed) Published
Abstract [en]

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 G(alpha i), 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.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC , 2019. Vol. 141, no 27, p. 10684-10701
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Biochemistry and Molecular Biology
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URN: urn:nbn:se:uu:diva-390792DOI: 10.1021/jacs.9b03193ISI: 000475533500017PubMedID: 31199130OAI: oai:DiVA.org:uu-390792DiVA, id: diva2:1343063
Funder
Knut and Alice Wallenberg Foundation, KAW 2013.0124Stiftelsen Olle Engkvist Byggmästare, 190-0335Wenner-Gren FoundationsGerman Research Foundation (DFG), 321722360Available from: 2019-08-15 Created: 2019-08-15 Last updated: 2019-08-15Bibliographically approved

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Calixto, Ana R.Moreira, CatiaPabis, AnnaKamerlin, Shina C. Lynn

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