GTP Hydrolysis Without an Active Site Base : A Unifying Mechanism for Ras and Related GTPases
Calixto, Ana R ; Moreira, Cátia ; Pabis, Anna ; Kötting, Carsten ; Gerwert, Klaus ; Rudack, Till and Kamerlin, Shina C L LU
(2019)
In Journal of the American Chemical Society
141(27).
p.10684-10701
- Abstract
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,... (More)
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.
(Less)
- author
- Calixto, Ana R
; Moreira, Cátia
; Pabis, Anna
; Kötting, Carsten
; Gerwert, Klaus
; Rudack, Till
and Kamerlin, Shina C L
LU
- publishing date
- 2019-07-10
- type
- Contribution to journal
- publication status
- published
- keywords
- Animals, Catalytic Domain, Enzyme Activation, GTP Phosphohydrolases/chemistry, Guanosine Triphosphate/metabolism, Humans, Hydrolysis, Models, Molecular
- in
- Journal of the American Chemical Society
- volume
- 141
- issue
- 27
- pages
- 18 pages
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- pmid:31199130
- scopus:85068309589
- ISSN
- 1520-5126
- DOI
- 10.1021/jacs.9b03193
- language
- English
- LU publication?
- no
- id
- 5bfa3b87-347c-4a1f-ad33-48c2f9b51a0c
- date added to LUP
- 2025-01-11 20:27:43
- date last changed
- 2025-07-13 18:43:45
@article{5bfa3b87-347c-4a1f-ad33-48c2f9b51a0c,
abstract = {{<p>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.</p>}},
author = {{Calixto, Ana R and Moreira, Cátia and Pabis, Anna and Kötting, Carsten and Gerwert, Klaus and Rudack, Till and Kamerlin, Shina C L}},
issn = {{1520-5126}},
keywords = {{Animals; Catalytic Domain; Enzyme Activation; GTP Phosphohydrolases/chemistry; Guanosine Triphosphate/metabolism; Humans; Hydrolysis; Models, Molecular}},
language = {{eng}},
month = {{07}},
number = {{27}},
pages = {{10684--10701}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Journal of the American Chemical Society}},
title = {{GTP Hydrolysis Without an Active Site Base : A Unifying Mechanism for Ras and Related GTPases}},
url = {{http://dx.doi.org/10.1021/jacs.9b03193}},
doi = {{10.1021/jacs.9b03193}},
volume = {{141}},
year = {{2019}},
}