Investigation of the active site of the cyanogenic β-D-glucosidase (Linamarase) from Manihot esculenta Crantz (Cassava). II. Identification of Glu-198 as an active site carboxylate group with acid catalytic function

Zsolt Keresztessy, Laszlo Kiss, Monica A. Hughes

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Abstract

The broad-specificity cyanogenic β-D-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crantz (cassava) was irreversibly inactivated by N-bromoacetyl-β-D-glucopyranosylamine according to pseudo-first-order kinetics with a second-order efficiency constant (ki/Ki = 0.1 min-1 M-1) identical for p-nitrophenyl-β-D-glucopyranosidase, p-nitrophenyl-β-D-galactopyranosidase, and linamarase activities of the enzyme. The competitive inhibitor p-nitrothiophenyl-β-D-glucopyranoside protected the enzyme from inactivation. pH dependence of the pseudo-first-order rate constant of inactivation revealed the involvement of an amino acid side chain in the inactivation process with pKa 7.0, which is very similar to that of the acid catalyst group of the enzyme (pKE2 = 7.2). The involved amino acid, which has to be ionized for the inactivation, was identified as Glu-198 using 14C-labeled inactivator to label the enzyme, cleaving the labeled protein into peptides and then purifying and sequencing the labeled peptide. This residue is highly conserved in the homologous family A β-glucosidases and family A1-A5 cellulases and lies in a consensus Asn-Glu-Pro motif occurring in all of these enzymes.

Original languageEnglish
Pages (from-to)323-330
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume315
Issue number2
DOIs
Publication statusPublished - dec. 1994

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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