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Titolo:
IMPORTANCE OF GLU-125 IN THE CATALYTIC ACTIVITY OF HUMAN RENAL DIPEPTIDASE
Autore:
ADACHI H; KATAYAMA T; NAKAZATO H; TSUJIMOTO M;
Indirizzi:
SUNTORY INST BIOMED RES SHIMAMOTO OSAKA 618 JAPAN SUNTORY INST BIOMED RES SHIMAMOTO OSAKA 618 JAPAN
Titolo Testata:
Biochimica et biophysica acta
fascicolo: 1, volume: 1163, anno: 1993,
pagine: 42 - 48
SICI:
0006-3002(1993)1163:1<42:IOGITC>2.0.ZU;2-4
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN MICROSOMAL DIPEPTIDASE; BETA-LACTAMASE ACTIVITY; DEHYDROPEPTIDASE-I; THIENAMYCIN; EXPRESSION; MEMBRANE; CLONING; SITE; PURIFICATION; SPECIFICITY;
Keywords:
RENAL DIPEPTIDASE; ACTIVE SITE; SITE-DIRECTED MUTAGENESIS; CHEMICAL MODIFICATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
29
Recensione:
Indirizzi per estratti:
Citazione:
H. Adachi et al., "IMPORTANCE OF GLU-125 IN THE CATALYTIC ACTIVITY OF HUMAN RENAL DIPEPTIDASE", Biochimica et biophysica acta, 1163(1), 1993, pp. 42-48

Abstract

The carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and dansylethylenediamine have shown to inhibit human renal dipeptidase (hrDP) irreversibly in a time-dependent manner. Cilastatin, a competitive inhibitor of the enzyme, partially protected the enzyme from inactivation. To identify the site(s) modified by EEDQ and dansylethylenediamine, the amino-acid sequence of tryptic fragments of modified enzyme were analyzed extensively. A comparison of the determined amino-acid sequences with the predicted primary structure of hrDP revealed that Glu-125 within the Glu115-Arg138 fragment was modified. In consequence, the role of Glu-125 in catalytic activity was investigated bysite-directed mutagenesis. Glu-125 was replaced by a glutamine, asparatic acid or cysteine residue. cDNAs for wild-type or mutated enzymes were expressed in CHO cells, and the resulting proteins were purified to apparent homogeneiety. The mutated enzyme, Gln-125-hrDP exhibited specific activity of 28.5 U/mg, corresponding to 11.4% of the wild-type. In contrast, Asp-125-hrDP and Cys-125hrDP were found to be inactive (less-than-or-equal-to 0.1% of wild-type enzyme). These results suggest that the polarity and/or length of the side chain of Glu-125 residueare important for the enzyme activity.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 29/11/20 alle ore 23:41:30