GTPase catalysis by Ras and other G-proteins: insights from Substrate Directed SuperImposition.

Comparisons of different protein structures are commonly carried out by superimposing the coordinates of the protein backbones or selected parts of the proteins. When the objective is analysis of similarities and differences in the enzyme's active site, there is an inherent problem in using the same domains for the superimposition. In this work we use a comparative approach termed here "Substrate Directed SuperImposition" (SDSI). It entails the superimposition of multiple protein-substrate structures using exclusively the coordinates of the comparable substrates. SDSI has the advantage of unbiased comparison of the active-site environment from the substrate's point of view. Our analysis extends previous usage of similar approaches to comparison of enzyme catalytic machineries. We applied SDSI to various G-protein structures for dissecting the mechanism of the GTPase reaction that controls the signaling activity of this important family. SDSI indicates that dissimilar G-proteins stabilize the transition state of the GTPase reaction similarly and supports the commonality of the critical step in this reaction, the reorientation of the critical arginine and glutamine. Additionally, we ascribe the catalytic inefficiency of the small G-protein Ras to the great flexibility of its active site and downplay the possible catalytic roles of the Lys16 residue in Ras GTPase. SDSI demonstrated that in contrast to all other Gly12 Ras mutants, which are oncogenic, the Gly12-->Pro mutant does not interfere with the catalytic orientation of the critical glutamine. This suggests why this mutant has a higher rate of GTP hydrolysis and is non-transforming. Remarkably, SDSI also revealed similarities in the divergent catalytic machineries of G-proteins and UMP/CMP kinase. Taken together, our results promote the use of SDSI to compare the catalytic machineries of both similar and different classes of enzymes.