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Titolo:
MECHANISTIC STUDIES ON THIAMINASE-I - OVEREXPRESSION AND IDENTIFICATION OF THE ACTIVE-SITE NUCLEOPHILE
Autore:
COSTELLO CA; KELLEHER NL; ABE M; MCLAFFERTY FW; BEGLEY TP;
Indirizzi:
CORNELL UNIV,BAKER LAB 120,DEPT CHEM ITHACA NY 14853 CORNELL UNIV,BAKER LAB 120,DEPT CHEM ITHACA NY 14853 YAMAGUCHI UNIV,SCH MED,DEPT MICROBIOL UBE YAMAGUCHI 755 JAPAN
Titolo Testata:
The Journal of biological chemistry
fascicolo: 7, volume: 271, anno: 1996,
pagine: 3445 - 3452
SICI:
0021-9258(1996)271:7<3445:MSOT-O>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
DISULFIDE BONDS; PROTEINS; CLEAVAGE; SEQUENCE; PEPTIDES; ANALOGS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
35
Recensione:
Indirizzi per estratti:
Citazione:
C.A. Costello et al., "MECHANISTIC STUDIES ON THIAMINASE-I - OVEREXPRESSION AND IDENTIFICATION OF THE ACTIVE-SITE NUCLEOPHILE", The Journal of biological chemistry, 271(7), 1996, pp. 3445-3452

Abstract

Thiaminase I (EC 2.5.1.2) catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles. Here we report the sequencing of a thiaminase I clone from Bacillus thiaminolyticus, the overexpression of the cloned gene in Escherichia coli, and the purification and characterization of the enzyme. Recombinant thiaminase I functions as a monomer with a K-m for thiamin of 3.7 +/- 0.6 mu M and a k(cat) of 34 s(-1). Electrospray ionization Fourier-transform mass spectrometry identified a single sequencing error and demonstrated heterogeneity, finding molecular weights of 42, 127, 42, 198, and 42,255 due to added Ala and Gly-Ala at the amino terminus. Similar analysis ofthe 4-amino-2-methyl-6-chloropyrimidine (8) inactivated enzyme indicated that the active site nucleophile involved in catalysis of the substitution reaction is located between Pro(79) and Thr(177). Subsequent cysteine-specific labeling and site-directed mutagenesis identified Cys(113) as the active site nucleophile.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 14/07/20 alle ore 19:49:24