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Titolo:
Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase - An essential residue in catalysis
Autore:
Cho, SW; Yoon, HY; Ahn, JY; Lee, EY; Lee, J;
Indirizzi:
Univ Ulsan, Coll Med, Dept Biochem, Seoul 138736, South Korea Univ Ulsan Seoul South Korea 138736 t Biochem, Seoul 138736, South Korea Yonsei Univ, Coll Hlth Sci, Dept Med Technol, Wonju, South Korea Yonsei Univ Wonju South Korea Sci, Dept Med Technol, Wonju, South Korea
Titolo Testata:
EUROPEAN JOURNAL OF BIOCHEMISTRY
fascicolo: 11, volume: 268, anno: 2001,
pagine: 3205 - 3213
SICI:
0014-2956(200106)268:11<3205:CMOL1O>2.0.ZU;2-3
Fonte:
ISI
Lingua:
ENG
Soggetto:
ESCHERICHIA-COLI; CLOSTRIDIUM-SYMBIOSUM; PYRIDOXAL 5'-PHOSPHATE; NEUROLOGICAL DISORDERS; NUCLEOTIDE-SEQUENCE; CRYSTAL-STRUCTURE; BOVINE BRAIN; BINDING-SITE; ISOPROTEINS; GENE;
Keywords:
glutamate dehydrogenase; gene synthesis; cassette mutagenesis; reactive lysine; pyridoxal 5 '-phosphate;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
43
Recensione:
Indirizzi per estratti:
Indirizzo: Cho, SW Univ Ulsan, Coll Med, Dept Biochem, 388-1 Poongnap Dong, Seoul 138736, South Korea Univ Ulsan 388-1 Poongnap Dong Seoul South Korea 138736 uth Korea
Citazione:
S.W. Cho et al., "Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase - An essential residue in catalysis", EUR J BIOCH, 268(11), 2001, pp. 3205-3213

Abstract

It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate duringcatalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5'-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despitean approximately 400-fold decrease in the respective apparent V-max of theLys130 mutant enzymes, apparent K-m values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-typeGDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 21/10/20 alle ore 10:41:43