RESCUE OF A MUTANT G-PROTEIN BY SUBSTRATE-ASSISTED CATALYSIS

Signaling by guanine-nucleotide-binding proteins (G-proteins) occurs w hen they are charged with GTP, while hydrolysis of the bound nucleotid e turns the signaling off. Despite a wealth of biochemical and structu ral information, the mechanism of GTP hydrolysis by G-proteins remains controversial. We have employed substrate-assisted catalysis as a nov el approach to study catalysis by G-proteins. In these studies. we hav e used diaminobenzophenone-phosphonoamidate-GTP, a unique GTP analog b earing the functional groups that are missing in the GTPase-deficient [Leu227]G(s alpha) mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP a nalog is hydrolyzed by this mutant and by the wild type enzyme at the same rate. On the other hand, modification of G(s alpha) by cholera to xin, which catalyses ADP-ribosylation of Arg201 of G(s alpha), decreas ed the rates of hydrolysis of both GTP and its analog by 95%. These re sults attest to the specificity of the GTP analog as a unique substrat e for the [Leu227]G(s alpha) mutant and to the essential role of Gln22 7 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild-type enzyme, favors a m odel in which formation of a pentavalent transition state intermediate , presumably stabilized by the catalytic glutamine, is not the rate-li miting step of the GTPase reaction.