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Titolo:
Consequences of a modified putative substrate-activation site on catalysisby yeast pyruvate decarboxylase
Autore:
Wang, J; Golbik, R; Seliger, B; Spinka, M; Tittmann, K; Hubner, G; Jordan, F;
Indirizzi:
Rutgers State Univ, Dept Chem, Newark, NJ 07102 USA Rutgers State Univ Newark NJ USA 07102 v, Dept Chem, Newark, NJ 07102 USA Rutgers State Univ, Program Cellular & Mol Biodynam, Newark, NJ 07102 USA Rutgers State Univ Newark NJ USA 07102 Mol Biodynam, Newark, NJ 07102 USA Univ Halle Wittenberg, Dept Biochem, Halle, Germany Univ Halle WittenbergHalle Germany nberg, Dept Biochem, Halle, Germany
Titolo Testata:
BIOCHEMISTRY
fascicolo: 6, volume: 40, anno: 2001,
pagine: 1755 - 1763
SICI:
0006-2960(20010213)40:6<1755:COAMPS>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
DIPHOSPHATE-DEPENDENT ENZYMES; THIAMIN DIPHOSPHATE; BREWERS-YEAST; ZYMOMONAS-MOBILIS; CRYSTAL-STRUCTURE; BENZOYLFORMATE DECARBOXYLASE; DIRECTED MUTAGENESIS; INFORMATION-TRANSFER; ANGSTROM RESOLUTION; MECHANISM;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
37
Recensione:
Indirizzi per estratti:
Indirizzo: Jordan, F Rutgers State Univ, Dept Chem, Newark, NJ 07102 USA Rutgers State Univ Newark NJ USA 07102 em, Newark, NJ 07102 USA
Citazione:
J. Wang et al., "Consequences of a modified putative substrate-activation site on catalysisby yeast pyruvate decarboxylase", BIOCHEM, 40(6), 2001, pp. 1755-1763

Abstract

Earlier, it had been proposed in the laboratories at Halle that a cysteineresidue is responsible for the hysteretic substrate activation behavior ofyeast pyruvate decarboxylase. More recently, this idea has received support in a series of studies from Rutgers with the identification of residue C221 as the site where substrate is bound to transmit the information to H92,to E91, to W412, and finally to the active center thiamin diphosphate. According to steady-state kinetic assays, the C221A/C222A variant is no longersubject to substrate activation yet is still a well-functioning enzyme. Several further experiments are reported on this variant: (I) The variant exhibits lag phases in the product formation progress curves, which can be attributed to a unimolecular step in the pre-steady-state stage of catalysis. (2) The rate of exchange with solvent deuterium of the thiamin diphosphate C2H atom is slowed by a factor of 2 compared to the wild-type enzyme, suggesting that the reduced activity that results from the substitutions some 20Angstrom from the active center is also seen in the first key step of the reaction. (3) The solvent (deuterium oxide) kinetic isotope effect was found to be inverse on V-max/K-m (0.62), and small but normal on V-max (1.26), virtually ruling out residue C221 as being responsible for the inverse effects reported for the wildtype enzyme at low substrate concentrations. The solvent kinetic isotope effects are compared to those on two related enzymesnot subject to substrate activation, Zymomonas mobilis pyruvate decarboxylase and benzoylformate decarboxylase.

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Documento generato il 28/03/20 alle ore 21:29:47