Quantitative exploration of the molecular origin of the activation of GTPase

Significance The origin of the activation of GTPases is explored considering the evidence that the transition state for the reference solution reaction involves a proton transfer between two water molecules. Ab initio quantum mechanical/molecular mechanical–based calibration of empirical valence bond surfaces of the solution reaction is used to simulate the GTPase activation process. The activation is found to reflect the same type of electrostatic stabilization obtained previously for the single water mechanism. The transition state proton transfer step does not appear to be rate limiting and thus is irrelevant to the catalytic effect. The calculated activation of RasGAP and EF-Tu reproduce the observed trend quantitatively and establishes its allosteric origin. We believe that our finding is general to all GTPases. GTPases play a major role in cellular processes, and gaining quantitative understanding of their activation demands reliable free energy surfaces of the relevant mechanistic paths in solution, as well as the interpolation of this information to GTPases. Recently, we generated ab initio quantum mechanical/molecular mechanical free energy surfaces for the hydrolysis of phosphate monoesters in solution, establishing quantitatively that the barrier for the reactions with a proton transfer (PT) step from a single attacking water (1W) is higher than the one where the PT is assisted by a second water (2W). The implication of this finding on the activation of GTPases is quantified here, by using the ab initio solution surfaces to calibrate empirical valence bond surfaces and then exploring the origin of the activation effect. It is found that, although the 2W PT path is a new element, this step is not rate determining, and the catalytic effect is actually due to the electrostatic stabilization of the pre-PT transition state and the subsequent plateau. Thus, the electrostatic catalytic effect found in our previous studies of the Ras GTPase activating protein (RasGAP) and the elongation factor-Tu (EF-Tu) with a 1W mechanism is still valid for the 2W path. Furthermore, as found before, the corresponding activation appears to involve a major allosteric effect. Overall, we believe that our finding is general to both GTPases and ATPases. In addition to the biologically relevant finding, we also provide a critical discussion of the requirements from reliable surfaces for enzymatic reactions.

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