Rescue of a mutant G-protein by substrate-assisted catalysis.
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]G(s alpha) 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 G(s alpha) by cholera toxin, which catalyses ADP-ribosylation of Arg201 of G(s alpha), 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]G(s alpha) 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.
Zor
T
T
Department of Biological Chemistry, the Kuhne Minerva Center for studies of visual transduction, The Hebrew University of Jerusalem, Israel.
Bar-Yaacov
M
M
Elgavish
S
S
Shaanan
B
B
Selinger
Z
Z
D000818:Animals; D001577:Benzophenones / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002772:Cholera Toxin / Q000494:pharmacology; D003198:Computer Simulation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D015398:Signal Transduction
C109051:3,4-diaminobenzophenone-phosphonoamidate-GTP; D001577:Benzophenones; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D019204:GTP-Binding Proteins