Analysis of a catalytic acidic pair in the active center of cellulase from Aspergillus aculeatus.
Biosci Biotechnol Biochem
; 63(12): 2157-62, 1999 Dec.
Article
en En
| MEDLINE
| ID: mdl-10664848
Four acidic amino acid residues, Asp97, Asp101, Glu118, and Glu202, were located in the cleft from the X-ray crystallographic analysis of FI-CMCase, endo-1,4-beta-glucanase (EC: 3.2.1.4) of Aspergillus aculeatus No. F-50. To identify the catalytic residues of the FI-CMCase, these residues were mutated to Glu or Ser from Asp97 and Asp101, and to Asp or Ser from Glu118 and Glu202 by site-directed mutagenesis, and totally 8 single mutant enzymes expressed in Escherichia coli were prepared: D97E, D97S, D101E, D101S, E118D, E118S, E202D, and E202S. Mutant enzymes E118S and E202S were not shown to have any detectable activity. Kinetic parameters of other mutant enzymes were measured after purification. The Km of mutant enzymes were not much different from that of wild type FI-CMCase, while the Vmax of mutant enzymes D97E, D97S, D101E, D101S, E118D, and D202E were much decreased to 1/50, 1/20, 1/4000, 1/2000, 1/800, and 1/1600 of the wild type FI-CMCase, respectively. From these results we concluded that Glu118 and Glu202 were most probable candidates for a catalytic pair of acidic amino acids in FI-CMCase.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Aspergillus
/
Celulasa
/
Ácido Glutámico
Idioma:
En
Revista:
Biosci Biotechnol Biochem
Asunto de la revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Año:
1999
Tipo del documento:
Article
País de afiliación:
Japón
Pais de publicación:
Reino Unido