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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Catalytic mechanism of inulinase from Arthrobacter sp. S37

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Kim, Kyoung-Yun ; Nascimento, Alessandro S. [2] ; Golubev, Alexander M. ; Polikarpov, Igor [4] ; Kim, Chung-Sei ; Kang, Su-Il ; Kim, Su-Il
Total Authors: 7
Document type: Journal article
Source: Biochemical and Biophysical Research Communications; v. 371, n. 4, p. 600-605, July 2008.
Field of knowledge: Biological Sciences - Biophysics

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)

FAPESP's process: 06/00182-8 - Structural biophysics of nuclear receptors and related proteins
Grantee:Igor Polikarpov
Support type: Research Projects - Thematic Grants