Rescue of a mutant G-protein by substrate-assisted catalysis

Tsaffrir Zor, Margalith Bar-Yaacov, Sharona Elgavish, Boaz Shaanan, Zvi Selinger*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations


Signaling by guanine-nucleotide-binding proteins (C-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 C-proteins. In these studies, we have used diaminobenzophenone-pho sphonoamidate-GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase-deficient [Leu227]G(sα) 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(sα) by cholera toxin, which catalyses ADP-ribosylation of Arg201 of G(sα), 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(sα) 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.

Original languageAmerican English
Pages (from-to)330-336
Number of pages7
JournalEuropean Journal of Biochemistry
Issue number1
StatePublished - 1997


  • GTP hydrolysis
  • GTP-binding protein
  • Substrate-assisted catalysis
  • cAMP


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