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Active site directed chemical modification of α-galactosidase from Bacillus stearothermophilus (NCIM 5146): Involvement of lysine, tryptophan and carboxylate residues in catalytic site

Paper ID Volume ID Publish Year Pages File Format Full-Text
18180 42713 2007 9 PDF Available
Title
Active site directed chemical modification of α-galactosidase from Bacillus stearothermophilus (NCIM 5146): Involvement of lysine, tryptophan and carboxylate residues in catalytic site
Abstract

The catalytic amino acid residues of the extracellular α-galactosidase (α-d-galactoside galactohydrolase; EC 3.2.1.22) from Bacillus stearothermophilus NCIM 5146 were investigated by pH dependence and chemical modification studies. These results suggested that carboxylate and a lysine residue take part in catalysis and only lysine residues were essential for substrate binding. Carbodiimide mediated chemical modification of the enzyme also supported that a carboxylate residue located in the active site act as a nucleophile base in substrate cleavage. Acylation and reductive methylation of lysine residues by acetic, citraconic anhydride and sodium borohydride suggested that four protonated lysine residues carrying positive charge on its ɛ-amino group provides the positive charge density for binding of the substrate. Additionally four tryptophan residues also found near to the active site and in a moderately hydrophobic environment. Kinetic and thermal inactivation study of modified enzyme indicated that these tryptophan residues might have a role in the catalytic site as well as in the thermal stabilization of active site conformation at higher temperature.

Keywords
Bacillus stearothermophilus; α-Galactosidase; Chemical modification; Tryptophan; Lysine; Carboxylate; Active site
First Page Preview
Active site directed chemical modification of α-galactosidase from Bacillus stearothermophilus (NCIM 5146): Involvement of lysine, tryptophan and carboxylate residues in catalytic site
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 5, 3 April 2007, Pages 1312–1320
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering