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How does GAP catalyze the GTPase reaction of Ras? A computer simulation study
T M Glennon1, J Villà, A Warshel
1Department of Chemistry, University of Southern California, Los Angeles, California 90089-1062, USA.
Biochemistry
|August 10, 2000
Summary
This study used computer simulations to investigate how GTPase-activating protein (GAP) accelerates the GTPase reaction of p21(Ras), a key protein in cell signaling and cancer development. Findings suggest GAP activates Ras through direct electrostatic interactions and indirect structural stabilization, clarifying Ras-Ras-GAP complex mechanisms.
Area of Science:
- Biochemistry and Molecular Biology
- Computational Chemistry
- Cell Signaling and Cancer Research
Background:
- The p21(Ras) protein regulates cell proliferation, and its GTPase reaction, accelerated by GTPase-activating protein (GAP), is crucial for signal termination.
- Understanding the mechanism of Ras-GAP interaction is vital for elucidating the role of Ras mutations in oncogenesis.
- Two primary hypotheses exist for GAP's catalytic effect: direct electrostatic interaction with the transition state or GAP-induced structural changes in Ras.
Purpose of the Study:
- To computationally investigate the relative importance of direct electrostatic interactions versus allosteric effects in GAP-mediated Ras activation.
- To explore the catalytic mechanisms of the Ras GTPase reaction, including associative and dissociative pathways.
- To examine the impact of specific Ras mutations, such as Gln61, on oncogenic potential within the Ras-GAP complex context.
Main Methods:
- Utilized the empirical valence bond (EVB) method for computer simulations of the Ras GTPase reaction.
- Simulated both associative and dissociative reaction pathways to analyze the catalytic mechanism.
- Employed coordinates of Ras from the Ras-GAP complex (Ras') to assess GAP's allosteric effects.
Main Results:
- Simulations accurately reproduced the experimentally observed catalytic effect of GAP (calculated: 7 ± 3 kcal/mol vs. observed: ~6.6 kcal/mol).
- The catalytic effect of Arg789 mutation was consistent with direct interaction with the transition state (calculated: 3-4 kcal/mol vs. observed: 4.5 kcal/mol).
- Simulations using Ras' coordinates revealed a significant catalytic effect, indicating GAP stabilizes a catalytic Ras configuration, favoring the GDP-bound state.
Conclusions:
- GAP activates Ras through a dual mechanism: direct electrostatic stabilization of the transition state and an indirect allosteric effect stabilizing the GDP-bound conformation.
- The oncogenic effect of Gln61 mutations appears indirect, likely due to structural changes induced by Ras-GAP complex formation rather than direct catalytic interference.
- The Ras GTPase reaction most likely proceeds via an associative mechanism.