Rescue of a Mutant G‐Protein by Substrate‐Assisted Catalysis

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Abstract
Signaling by guanine‐nucleotide‐binding proteins (G‐proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G‐proteins remains controversial. We have employed substrate‐assisted catalysis as a novel approach to study catalysis by G‐proteins. In these studies, we have used diaminobenzophenone‐phosphonoamidate‐GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase‐deficient [Leu227]Gsα mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild‐type enzyme at the same rate. On the other hand, modification of Gsα by cholera toxin, which catalyses ADP‐ribosylation of Arg201 of Gsα, decreased the rates of hydrolysis of both GTP and its analog by 95%. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]Gsα mutant and to the essential role of Gln227 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 model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the rate‐limiting step of the GTPase reaction.