Transition-state structure for the ADP-ribosylation of recombinant G(iα1) subunits by pertussis toxin

Johannes Scheuring, Paul J. Berti, Vern L. Schramm

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Pertussis toxin ADP-ribosylates a specific Cys side chain in the α- subunit of several G-proteins. Recombinant G(iα1)-subunits were rapidly ADP- ribosylated in the absence of βγ-subunits, with a K(m) of 800 μM and a k(cat) of 40 min-1. Addition of βγ-subunits decreases K(m) to 0.3 μM with little change of k(cat). Kinetic isotope effects established the transition-state structure for ADP-ribosylation of G(iα1) subunits. The transition state is dissociative, with a 2.1 Å bond to the nicotinamide leaving group and a bond of 2.5 Å to the sulfur nucleophile. The nucleophilic participation of G(iα1) at the transition state is greater than that for water in the hydrolysis of NAD+ by pertussis toxin. Crystal structures for G(iα1) show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered G(iα1) conformation to expose and ionize Cys. The transition state has been docked into the crystal structure of pertussis toxin in a geometry required for transition state formation.

Original languageEnglish (US)
Pages (from-to)2748-2758
Number of pages11
JournalBiochemistry
Volume37
Issue number9
DOIs
StatePublished - Mar 3 1998

Fingerprint

Pertussis Toxin
Adenosine Diphosphate
Nucleophiles
NAD
Crystal structure
Niacinamide
GTP-Binding Proteins
Sulfur
Isotopes
Conformations
Hydrolysis
Kinetics
Geometry
Water

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transition-state structure for the ADP-ribosylation of recombinant G(iα1) subunits by pertussis toxin. / Scheuring, Johannes; Berti, Paul J.; Schramm, Vern L.

In: Biochemistry, Vol. 37, No. 9, 03.03.1998, p. 2748-2758.

Research output: Contribution to journalArticle

@article{221be3777b244f479b45107c194c3b21,
title = "Transition-state structure for the ADP-ribosylation of recombinant G(iα1) subunits by pertussis toxin",
abstract = "Pertussis toxin ADP-ribosylates a specific Cys side chain in the α- subunit of several G-proteins. Recombinant G(iα1)-subunits were rapidly ADP- ribosylated in the absence of βγ-subunits, with a K(m) of 800 μM and a k(cat) of 40 min-1. Addition of βγ-subunits decreases K(m) to 0.3 μM with little change of k(cat). Kinetic isotope effects established the transition-state structure for ADP-ribosylation of G(iα1) subunits. The transition state is dissociative, with a 2.1 {\AA} bond to the nicotinamide leaving group and a bond of 2.5 {\AA} to the sulfur nucleophile. The nucleophilic participation of G(iα1) at the transition state is greater than that for water in the hydrolysis of NAD+ by pertussis toxin. Crystal structures for G(iα1) show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered G(iα1) conformation to expose and ionize Cys. The transition state has been docked into the crystal structure of pertussis toxin in a geometry required for transition state formation.",
author = "Johannes Scheuring and Berti, {Paul J.} and Schramm, {Vern L.}",
year = "1998",
month = "3",
day = "3",
doi = "10.1021/bi972594x",
language = "English (US)",
volume = "37",
pages = "2748--2758",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "9",

}

TY - JOUR

T1 - Transition-state structure for the ADP-ribosylation of recombinant G(iα1) subunits by pertussis toxin

AU - Scheuring, Johannes

AU - Berti, Paul J.

AU - Schramm, Vern L.

PY - 1998/3/3

Y1 - 1998/3/3

N2 - Pertussis toxin ADP-ribosylates a specific Cys side chain in the α- subunit of several G-proteins. Recombinant G(iα1)-subunits were rapidly ADP- ribosylated in the absence of βγ-subunits, with a K(m) of 800 μM and a k(cat) of 40 min-1. Addition of βγ-subunits decreases K(m) to 0.3 μM with little change of k(cat). Kinetic isotope effects established the transition-state structure for ADP-ribosylation of G(iα1) subunits. The transition state is dissociative, with a 2.1 Å bond to the nicotinamide leaving group and a bond of 2.5 Å to the sulfur nucleophile. The nucleophilic participation of G(iα1) at the transition state is greater than that for water in the hydrolysis of NAD+ by pertussis toxin. Crystal structures for G(iα1) show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered G(iα1) conformation to expose and ionize Cys. The transition state has been docked into the crystal structure of pertussis toxin in a geometry required for transition state formation.

AB - Pertussis toxin ADP-ribosylates a specific Cys side chain in the α- subunit of several G-proteins. Recombinant G(iα1)-subunits were rapidly ADP- ribosylated in the absence of βγ-subunits, with a K(m) of 800 μM and a k(cat) of 40 min-1. Addition of βγ-subunits decreases K(m) to 0.3 μM with little change of k(cat). Kinetic isotope effects established the transition-state structure for ADP-ribosylation of G(iα1) subunits. The transition state is dissociative, with a 2.1 Å bond to the nicotinamide leaving group and a bond of 2.5 Å to the sulfur nucleophile. The nucleophilic participation of G(iα1) at the transition state is greater than that for water in the hydrolysis of NAD+ by pertussis toxin. Crystal structures for G(iα1) show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered G(iα1) conformation to expose and ionize Cys. The transition state has been docked into the crystal structure of pertussis toxin in a geometry required for transition state formation.

UR - http://www.scopus.com/inward/record.url?scp=0032478328&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032478328&partnerID=8YFLogxK

U2 - 10.1021/bi972594x

DO - 10.1021/bi972594x

M3 - Article

VL - 37

SP - 2748

EP - 2758

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 9

ER -