Abstract
We present a systematic evaluation of different possible reaction mechanisms for GTP hydrolysis in RhoA, a member of the Ras superfamily of enzymes that uses this reaction to switch from an active to an inactive conformation. These enzymes are activated by the presence of a GTPase activating protein (or GAP) that forms an intimate complex with residues of the two proteins present in the active site. We have explored the multidimensional reactional free energy landscape in the active site of the complex formed by RhoA and p50RhoGAP. Our molecular dynamics simulations show that the activating enzyme p50RhoGAP establishes catalytically important interactions with the phosphate groups of GTP through its so-called arginine finger (Arg85) and also with the RhoA residue Gln63. This is a key residue because it not only interacts with the nucleophilic water molecule but also participates actively in the reaction mechanism. Adaptive string method simulations using hybrid quantum mechanics/molecular mechanics (QM/MM) potentials with both tight-binding and density functional Hamiltonians show that GTP hydrolysis proceeds through the formation of a metaphosphate metastable species. Mechanistic proposals differ in the proton transfer rearrangements required to form the inorganic phosphate ion. Our simulations discard a solvent-assisted mechanism and point to the participation of Gln63 in the proton transfer process by means of the side chain tautomerism from the amide to the imide form. The proton transfer required to recover the amide form of Gln63 requires the participation of the inorganic phosphate, and it is the rate-limiting step of the process, with a free energy barrier of 20.2 kcal mol(-1) at the B3LYPD3/MM level, in good agreement with the experimentally derived value. The amide-imide tautomerism could also be relevant in other enzymes, facilitating proton transfer events in complex mechanisms.