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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

Catalytic mechanism of inulinase from Arthrobacter sp. S37

Texto completo
Autor(es):
Kim, Kyoung-Yun ; Nascimento, Alessandro S. [2] ; Golubev, Alexander M. ; Polikarpov, Igor [4] ; Kim, Chung-Sei ; Kang, Su-Il ; Kim, Su-Il
Número total de Autores: 7
Tipo de documento: Artigo Científico
Fonte: Biochemical and Biophysical Research Communications; v. 371, n. 4, p. 600-605, July 2008.
Área do conhecimento: Ciências Biológicas - Biofísica
Assunto(s):Biofísica   Catalisadores   Bactérias   Arthrobacter   Mutagênese sítio-dirigida   Ácido glutâmico   Ácido aspártico
Resumo

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (kcat/Km) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in kcat, but not due to variations in Km, consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pKa of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pKa. Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme. (AU)

Processo FAPESP: 04/08070-9 - Estudos estruturais do receptor de mineralocorticóide humano
Beneficiário:Alessandro Silva Nascimento
Linha de fomento: Bolsas no Brasil - Doutorado Direto
Processo FAPESP: 06/00182-8 - Biofísica estrutural dos receptores nucleares e proteínas relacionadas
Beneficiário:Igor Polikarpov
Linha de fomento: Auxílio à Pesquisa - Temático