Transition state studies on the adp-ribosylation of g-proteins catalyzed by pertussis toxin

J. Scheuring, V. L. Schramm

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Abstract

Pertussis Toxin (PTX) ADP-ribosylates a cysteine residue at the o-bubunits from several G-proteins. using NAD+ as a substrate. In absence of an ;tc ceptor, PTX acts as a slow NAD-glycohydrolase. Isolated o-subunits from the trimeric Cî-proteins arc reported to be weak acceptors for ADP-rihosylation by PTX. Using recombinant G,] o subunits at concentrations up to '2 m M made it possible to obtain high rates of ADP-ribosvlation, sufficient for mechanistic studies. \KM of O.S mM and a kca( of 40 min"1 were found. PTX is a target for inhibitors based on the transition state structure. Kinetic isotope effects were determined using labeled NAD"1" for both the ADP-ribosylation and the hydrolysis reaction, and models of the transition state structures were derived by normal bond vibrational analysis of these data. The transition states are characterized bv a partially dissociated nicotinamide group and a oxocarbeniunvion like structure of the ribose ring. There is onfy weak interaction to the water nucleophile in the hydrolysis, but significant participation of the sulfur atom in the ADP-ribosylation reaction. In the latter, inverse solvent isotope effects indicated deprotonation prior to transition state formation, the thiolate anion may stabilize the developing positive charge in the ribose ring. NAD+ analogues with the nicotinamide ribose replaced by an iminoribitoi ring are expected to be potent transition state inhibitors. This work was support.

Original languageEnglish (US)
Pages (from-to)A1034
JournalFASEB Journal
Volume11
Issue number9
Publication statusPublished - Dec 1 1997

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ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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