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.D etailed understanding of many problems in biology boils down to the elucidation of the correct reaction mechanism in the protein and in solution and to the elucidation of the origin of the corresponding catalytic effect. However, this requires overcoming the challenge of obtaining accurate free energy surfaces in the condensed phase. Such a task can be accomplished, in principle, by the combined quantum mechanical/molecular mechanical (QM/ MM) method (1-5). However, the implementation of this method is still very challenging. At present, we are not at the stage reached in studies of gas phase reactions, where the results of ab initio calculations are sometimes considered almost as substitutes for experimental results. Nevertheless, recent advances in ab initio QM/MM [QM(ai)/MM] studies (2, 4-6) brought us closer to having quantitative results for solution reactions and in some respects to quantitative results for enzymes, especially if one evaluates reliable QM (ai)/MM free energy surfaces for the solution reaction and then use the resulting surfaces to calibrate empirical valence bond (EVB) surfaces for studying the relevant enzymatic reaction. Here we exploit these advances by exploring the hydrolysis of phosphate monoesters that is the key to the activation of GTPases. That is, we start by quantifying the energetics of the solution reaction and then use the solution free energy surface to calibrate EVB surface that allow us to reliably...

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