Pertussis toxin: Transition state analysis for ADP-ribosylation of G- protein peptide α(i3)C20

Johannes Scheuring, Vern L. Schramm

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Abstract

Pertussis toxin from Bordetella pertussis is one of the ADP- ribosylating toxins which are the cytotoxic agents of several infectious diseases. Transition state analogues of these enzymes are expected to be potent inhibitors and may be useful in therapy. Pertussis toxin catalyzes the ADP-ribosylation of a cysteine in the synthetic peptide α(i3)C20, corresponding to the C-terminal 20 amino acids of the α-subunits of the G- protein G(i3). A family of kinetic isotope effects was determined for the ADP-ribosylation reaction, using 3H-, 14C- and 15N-labeled NAD+ as substrates. Primary kinetic isotope effects were 1.050 ± 0.006 for [1'(N)- 14C] and 1.021 ± 0.002 for [1(N)-15N], the double primary effect of [1'(N)-14C, 1(N)-15N] was 1.064 ± 0.002. Secondary kinetic isotope effects were 1.208 ± 0.014 for [1'(N)-3H], 1.104 ± 0.010 for [2'(N)-3H], 0.989 ± 0.001 for [4'(N)-3H], and 1.014 ± 0.002 for [5'(N)-3H]. Isotope trapping experiments yielded a commitment factor of 0.01, demonstrating that the observed isotope effects are near intrinsic. Solvent D2O kinetic isotope effects are inverse, consistent with deprotonation of the attacking Cys prior to transition state formation. The transition state structure was determined by a normal mode bond vibrational analysis. The transition state is characterized by a nicotinamide leaving group bond order of 0.14, corresponding to a bond length of 2.06 Å. The incoming thiolate nucleophile has a bond order of 0.11, corresponding to 2.47 Å. The ribose ring has strong oxocarbenium ion character. Pertussis toxin also catalyzes the slow hydrolysis of NAD+ in the absence of peptides. Comparison of the transition states for NAD+ hydrolysis and for ADP-ribosylation of peptide α(i3)C20 indicates that the sulfur nucleophile from the peptide Cys participates more actively as a nucleophile in the reaction than does water in the hydrolytic reaction. Participation of the thiolate anion at the transition state provides partial neutralization of the cationic charge which normally develops at the transition states of N-ribohydrolases and transferases. Thus, the presence of the peptide provides increased S(N2) character in a loose transition state which retains oxocarbenium character in the ribose.

Original languageEnglish (US)
Pages (from-to)8215-8223
Number of pages9
JournalBiochemistry
Volume36
Issue number27
DOIs
StatePublished - Jul 8 1997

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Pertussis Toxin
GTP-Binding Proteins
Isotopes
Adenosine Diphosphate
Peptides
Nucleophiles
NAD
Kinetics
Ribose
Hydrolysis
Bordetella pertussis
Deprotonation
Niacinamide
Cytotoxins
Bond length
Transferases
Sulfur
Anions
Communicable Diseases
Cysteine

ASJC Scopus subject areas

  • Biochemistry

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Pertussis toxin : Transition state analysis for ADP-ribosylation of G- protein peptide α(i3)C20. / Scheuring, Johannes; Schramm, Vern L.

In: Biochemistry, Vol. 36, No. 27, 08.07.1997, p. 8215-8223.

Research output: Contribution to journalArticle

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title = "Pertussis toxin: Transition state analysis for ADP-ribosylation of G- protein peptide α(i3)C20",
abstract = "Pertussis toxin from Bordetella pertussis is one of the ADP- ribosylating toxins which are the cytotoxic agents of several infectious diseases. Transition state analogues of these enzymes are expected to be potent inhibitors and may be useful in therapy. Pertussis toxin catalyzes the ADP-ribosylation of a cysteine in the synthetic peptide α(i3)C20, corresponding to the C-terminal 20 amino acids of the α-subunits of the G- protein G(i3). A family of kinetic isotope effects was determined for the ADP-ribosylation reaction, using 3H-, 14C- and 15N-labeled NAD+ as substrates. Primary kinetic isotope effects were 1.050 ± 0.006 for [1'(N)- 14C] and 1.021 ± 0.002 for [1(N)-15N], the double primary effect of [1'(N)-14C, 1(N)-15N] was 1.064 ± 0.002. Secondary kinetic isotope effects were 1.208 ± 0.014 for [1'(N)-3H], 1.104 ± 0.010 for [2'(N)-3H], 0.989 ± 0.001 for [4'(N)-3H], and 1.014 ± 0.002 for [5'(N)-3H]. Isotope trapping experiments yielded a commitment factor of 0.01, demonstrating that the observed isotope effects are near intrinsic. Solvent D2O kinetic isotope effects are inverse, consistent with deprotonation of the attacking Cys prior to transition state formation. The transition state structure was determined by a normal mode bond vibrational analysis. The transition state is characterized by a nicotinamide leaving group bond order of 0.14, corresponding to a bond length of 2.06 {\AA}. The incoming thiolate nucleophile has a bond order of 0.11, corresponding to 2.47 {\AA}. The ribose ring has strong oxocarbenium ion character. Pertussis toxin also catalyzes the slow hydrolysis of NAD+ in the absence of peptides. Comparison of the transition states for NAD+ hydrolysis and for ADP-ribosylation of peptide α(i3)C20 indicates that the sulfur nucleophile from the peptide Cys participates more actively as a nucleophile in the reaction than does water in the hydrolytic reaction. Participation of the thiolate anion at the transition state provides partial neutralization of the cationic charge which normally develops at the transition states of N-ribohydrolases and transferases. Thus, the presence of the peptide provides increased S(N2) character in a loose transition state which retains oxocarbenium character in the ribose.",
author = "Johannes Scheuring and Schramm, {Vern L.}",
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T2 - Transition state analysis for ADP-ribosylation of G- protein peptide α(i3)C20

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N2 - Pertussis toxin from Bordetella pertussis is one of the ADP- ribosylating toxins which are the cytotoxic agents of several infectious diseases. Transition state analogues of these enzymes are expected to be potent inhibitors and may be useful in therapy. Pertussis toxin catalyzes the ADP-ribosylation of a cysteine in the synthetic peptide α(i3)C20, corresponding to the C-terminal 20 amino acids of the α-subunits of the G- protein G(i3). A family of kinetic isotope effects was determined for the ADP-ribosylation reaction, using 3H-, 14C- and 15N-labeled NAD+ as substrates. Primary kinetic isotope effects were 1.050 ± 0.006 for [1'(N)- 14C] and 1.021 ± 0.002 for [1(N)-15N], the double primary effect of [1'(N)-14C, 1(N)-15N] was 1.064 ± 0.002. Secondary kinetic isotope effects were 1.208 ± 0.014 for [1'(N)-3H], 1.104 ± 0.010 for [2'(N)-3H], 0.989 ± 0.001 for [4'(N)-3H], and 1.014 ± 0.002 for [5'(N)-3H]. Isotope trapping experiments yielded a commitment factor of 0.01, demonstrating that the observed isotope effects are near intrinsic. Solvent D2O kinetic isotope effects are inverse, consistent with deprotonation of the attacking Cys prior to transition state formation. The transition state structure was determined by a normal mode bond vibrational analysis. The transition state is characterized by a nicotinamide leaving group bond order of 0.14, corresponding to a bond length of 2.06 Å. The incoming thiolate nucleophile has a bond order of 0.11, corresponding to 2.47 Å. The ribose ring has strong oxocarbenium ion character. Pertussis toxin also catalyzes the slow hydrolysis of NAD+ in the absence of peptides. Comparison of the transition states for NAD+ hydrolysis and for ADP-ribosylation of peptide α(i3)C20 indicates that the sulfur nucleophile from the peptide Cys participates more actively as a nucleophile in the reaction than does water in the hydrolytic reaction. Participation of the thiolate anion at the transition state provides partial neutralization of the cationic charge which normally develops at the transition states of N-ribohydrolases and transferases. Thus, the presence of the peptide provides increased S(N2) character in a loose transition state which retains oxocarbenium character in the ribose.

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