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Titolo:
PERTUSSIS TOXIN - TRANSITION-STATE ANALYSIS FOR ADP-RIBOSYLATION OF G-PROTEIN PEPTIDE ALPHA(I3)C20
Autore:
SCHEURING J; SCHRAMM VL;
Indirizzi:
YESHIVA UNIV ALBERT EINSTEIN COLL MED,DEPT BIOCHEM,1300 MORRIS PK AVEBRONX NY 10461 YESHIVA UNIV ALBERT EINSTEIN COLL MED,DEPT BIOCHEM BRONX NY 10461
Titolo Testata:
Biochemistry
fascicolo: 27, volume: 36, anno: 1997,
pagine: 8215 - 8223
SICI:
0006-2960(1997)36:27<8215:PT-TAF>2.0.ZU;2-#
Fonte:
ISI
Lingua:
ENG
Soggetto:
PURINE NUCLEOSIDE PHOSPHORYLASE; ISLET-ACTIVATING PROTEIN; GTP-BINDING PROTEINS; CATALYZED REACTION; AMP NUCLEOSIDASE; ALPHA-SUBUNITS; BETA-GAMMA; HYDROLYSIS; MECHANISM; INHIBITORS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
49
Recensione:
Indirizzi per estratti:
Citazione:
J. Scheuring e V.L. Schramm, "PERTUSSIS TOXIN - TRANSITION-STATE ANALYSIS FOR ADP-RIBOSYLATION OF G-PROTEIN PEPTIDE ALPHA(I3)C20", Biochemistry, 36(27), 1997, pp. 8215-8223

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 catalyzesthe ADP-ribosylation of a cysteine in the synthetic peptide alpha(i3)C20, corresponding to the C-terminal 20 amino acids of the ol-subunitsof the G-protein G(i3). A family of kinetic isotope effects was determined for the ADP-ribosylation reaction, using H-3-, C-14 (-) and N-15-labeled NAD(+) as substrate. Primary kinetic isotope effects were 1.050 +/- 0.006 for [1'(N)-C-14] and 1.021 +/- 0.002 for [1(N)-N-15], thedouble primary effect of [1'(N)-C-14,1(N)-N-15] was 1.064 +/- 0.002. Secondary kinetic isotope effects were 1.208 +/- 0.014 for [1'(N)-H-3], 1.104 +/- 0.010 for [2'(N)-H-3], 0.989 1:+/- 0.001 for [4'(N)-H-3], and 1.014 +/- 0.002 for [5'(N)-H-3]. Isotope trapping experiments yielded a commitment factor of 0.01, demonstrating that the observed isotope effects are near intrinsic. Solvent D2O kinetic isotope effects areinverse, 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 Angstrom. The incoming thiolate nucleophile has a bond order of 0.11, corresponding to 2.47 Angstrom. 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 alpha(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 al 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(N)2 character in a loose transition state which retains oxocarbenium character in the ribose.

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Documento generato il 18/09/20 alle ore 10:55:49