Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

Abstract

Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach Ras and other G proteins are active in signaling when GTP is bound. Hydrolysis of GTP to give bound GDP turns these signaling proteins off (1-3). For this reason, the mechanism of GTP hydrolysis by Ras and other G proteins has elicited much interest. Nevertheless, despite a wealth of structural, kinetic, and site-directed mutagenesis data, the catalytic mechanism of Ras remains controversial.Crucial to an understanding of enzymatic catalysis is knowledge of the nature of the transition state of the reaction and how that transition state differs from the ground state; transition state theory defines catalysis as stabilization of a reaction's transition state relative to its ground state. An underlying feature comrmon among the several distinct models presented for Ras catalysis is an explicit or implicit assumption that the transition state is associative in character. However, recent model studies have shown that the transition state for GTP hydrolysis in solution is dissociative rather than associative (4). This transition state information is used to evaluate previously proposed mechanisms from a chemical perspective.A new catalytic mechanism is then proposed, involving a hydrogen bond to the ,3-y bridge oxygen of GTP. This proposal is consistent with pre-existing structural, spectral, and energetic data.

Why nature really chose phosphate

“…It seems to us that the above misunderstanding is compounded by the recent argument (Lassila et al 2011) that (Maegley et al 1996) actually predicted the effect of the Arg finger (Fig. 27) and its relationship to the dissociative mechanism (now suddenly called ' loose ' TS).…”

“…Here we have a problematic assertion that overlooks the fact that this is not the relevant issue. The problem (which originated in, Maegley et al 1996) is associated with the confusion between the uncatalyzed reaction for a given path (for instance concerted PT to the non-bridging oxygen on the phosphate) and the catalysis of the given mechanism (pathway) by the change in environment, where it is basically argued that this path with a PT provides no catalytic advantage, However, the catalysis is not due to the PT (this is a mechanism with its intrinsic energy is the energy of the reference reaction), but rather to the change of the energy of this mechanism due to the environment. The actual catalysis comes from the enzyme preorganization, including the electrostatic effect of the Mg 2+ ion, and not from the specific mechanism used (the same mechanism used by the enzyme is much slower than in water ; see Warshel et al 2006b).…”

“…Related problematic arguments were also used in the claim (Maegley et al 1996) that the presumed charge buildup on the c-phosphate in a dissociative TS means that there is no stabilizing interaction between the external charge and the non-bridging oxygens of c-phosphate (i.e. that the catalytic effect of the arginine is limited to interactions with the leaving group oxygen), and that interactions with the c-phosphate would only become important if the enzyme caused a switch to an associative TS.…”

“…Apparently (see above) it was suggested that a dissociative TS cannot be stabilize by positive charge, stating that ' Although electrostatic interactions with the c-phosphoryl oxygens are not predicted to be strengthened in a dissociative TS and may even be somewhat weakened, such interactions may be present because they are important for binding and positioning. ' In fact (Maegley et al 1996) completely overlooked the crucial The free-energy profiles of the GTPase reaction in the native RasGAP complex (stars) and its different mutations, R789R NP (squares), Q61Q NP (triangles) and Q61L (circles). The profiles were obtained from an average over five different profiles, calculated using the EVB FEP/Umbrella sampling procedure.…”

“…So, clearly, the proton has to be transferred somewhere, and will not remain on the water molecule for the entire duration of the reaction (as this results in an energetically unfavorable species). Maegley et al 1996 argued that, in the TS, the proton will have almost fully been transferred to the leaving group. However, the proton cannot fly over the phosphate from the water molecule to the leaving group, so, if leaving group protonation is even at all required, some form of proton relay is required.…”