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

J. Scheuring, Vern L. Schramm

Research output: Contribution to journalArticle

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)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997

Fingerprint

pertussis toxin
Pertussis Toxin
Adenosine Diphosphate
ribose
nicotinamide
NAD
Ribose
isotopes
Proteins
proteins
Isotopes
hydrolysis
Hydrolysis
NAD+ Nucleosidase
glycosidases
G-proteins
Deprotonation
Nucleophiles
Niacinamide
anions

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Transition state studies on the adp-ribosylation of g-proteins catalyzed by pertussis toxin. / Scheuring, J.; Schramm, Vern L.

In: FASEB Journal, Vol. 11, No. 9, 1997.

Research output: Contribution to journalArticle

@article{a46f8ee43ac443248f6ec7af6989da65,
title = "Transition state studies on the adp-ribosylation of g-proteins catalyzed by pertussis toxin",
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 C{\^i}-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.",
author = "J. Scheuring and Schramm, {Vern L.}",
year = "1997",
language = "English (US)",
volume = "11",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "9",

}

TY - JOUR

T1 - Transition state studies on the adp-ribosylation of g-proteins catalyzed by pertussis toxin

AU - Scheuring, J.

AU - Schramm, Vern L.

PY - 1997

Y1 - 1997

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33750186700&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33750186700&partnerID=8YFLogxK

M3 - Article

VL - 11

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 9

ER -