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Titolo:
INVESTIGATION OF A CATALYTIC ZINC-BINDING SITE IN ESCHERICHIA-COLI L-THREONINE DEHYDROGENASE BY SITE-DIRECTED MUTAGENESIS OF CYSTEINE-38
Autore:
JOHNSON AR; CHEN YW; DEKKER EE;
Indirizzi:
UNIV MICHIGAN,DEPT BIOL CHEM ANN ARBOR MI 48109 UNIV MICHIGAN,DEPT BIOL CHEM ANN ARBOR MI 48109
Titolo Testata:
Archives of biochemistry and biophysics (Print)
fascicolo: 2, volume: 358, anno: 1998,
pagine: 211 - 221
SICI:
0003-9861(1998)358:2<211:IOACZS>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
YEAST ALCOHOL-DEHYDROGENASE; SORBITOL DEHYDROGENASE; CARBOXYL GROUPS; ACTIVATION; K-12; DNA; IDENTIFICATION; PURIFICATION; RESIDUE; ENZYME;
Keywords:
THREONINE DEHYDROGENASE; SITE-DIRECTED MUTAGENESIS; PROTEIN ISOLATION AND PURIFICATION; ZINC ENZYME; ENZYME ACTIVE SITE; METAL ION ACTIVATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
33
Recensione:
Indirizzi per estratti:
Citazione:
A.R. Johnson et al., "INVESTIGATION OF A CATALYTIC ZINC-BINDING SITE IN ESCHERICHIA-COLI L-THREONINE DEHYDROGENASE BY SITE-DIRECTED MUTAGENESIS OF CYSTEINE-38", Archives of biochemistry and biophysics (Print), 358(2), 1998, pp. 211-221

Abstract

L-Threonine dehydrogenase catalyzes the NAD(+)- dependent oxidation of threonine forming 2-amino-3-ketobutyrate. Chemical modification of Cys-38 of Escherichia coil threonine dehydrogenase, whose residue aligns with the catalytic zinc-binding residue, Cys-46, of related alcohol/polyol dehydrogenases, inactivates the enzyme [B. R. Epperly and E. E. Dekker (1991) J. Biol. Chem. 266, 6086-6092; A. R. Johnson and E. E. Dekker (1996) Protein Sci. 5, 382-390]. To probe its function, Cys-38 was changed to Ser, Asp, and Glu by site-directed mutagenesis. MutantsC38S and C38D were purified to homogeneity and found to be, like the wild-type enzyme, homotetrameric proteins containing one Zn2+ atom persubunit. The circular dichroism spectra of these mutants were essentially identical to that of the wild-type enzyme. Mutant C38S was catalytically inactive but mutant C38D had a specific activity of 0.2 unit/mg, a level similar to 1% that of the wild-type enzyme. After it was incubated with 1 mM Zn2+ and then assayed in the presence of 15 mM Zn2+,mutant C38S showed only a trace of enzymatic activity (i.e., 0.013 unit/mg). Preincubation of mutant C38D with 5 mM Zn2+, Co2+, Or Cd2+ increased its activity 57-, 6-, or 3-fold, respectively; 1 mM Mn2+ halvedand 0.5 mM Hg2+ abolished activity. Zn2+-stimulated mutant C38D showed these properties: apparent substrate activation at low threonine concentrations, a maximum activity of 27 units/mg with 20 mM threonine, and inhibition by high levels of substrate; an activation K-d = 3 mM Zn2+; and a pH optimum of 8.4 (in contrast to pH 10.3 for the wild-type enzyme). Without added Zn2+, mutant C38D is equally active with threonine and 2-amino-3-hydroxypentanoate, but Zn2+-activated mutant C38D is10-fold more reactive with threonine than with 8-amino3-hydroxypentanoate, In the absence of added metal ions, wild-type enzyme similarly uses substrates other than threonine and shows a dramatic increase in activity with only threonine when stimulated by either Cd2+ or Mn2+; added Zn2+ has no effect on activity with threonine. Cys-38 of threoninedehydrogenase, therefore, is located in an activating divalent metal ion-binding site. Having a negatively charged residue like Asp in thisposition allows the binding of a catalytic Zn2+ ion which enhances activity with threonine and reduces activity with substrate analogs. Whether Cys-38 of wild-type threonine dehydrogenase binds a catalytic metal ion (possibly Zn2+) in vivo remains to be established. (C) 1998 Academic Press.

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Documento generato il 26/11/20 alle ore 10:26:17