Catalytic mechanism of inulinase from Arthrobacter sp. S37

Author(s)

Kim, Kyoung-Yun ; Nascimento, Alessandro S. ^[2] ; Golubev, Alexander M. ; Polikarpov, Igor ^[4] ; Kim, Chung-Sei ; Kang, Su-Il ; Kim, Su-Il

• 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

Abstract

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