Investigation of the active site of the extracellular β-D-glucosidase from Aspergillus carbonarius

Szilvia Jäger, László Kiss

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5 Citations (Scopus)

Abstract

The catalytic amino acid residues of the extracellular β-D-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus carbonarius were investigated. The pH dependence curves gave apparent pK values of 2.8 and 5.93 for the free enzyme, and 2.24 and 6.14 for the enzyme-substrate complex using p-nitrophenyl-β-D-glucoside as substrate. Carbodiimide- and Woodward reagent K-mediated chemical modifications suggested that a carboxylate residue, located in the active centre, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.61 in the modification reaction, proving that a carboxylate residue was modified. The A. carbonarius β-glucosidase was irreversibly inactivated by N-bromoacetyl-β-D-glucopyranosylamine. The active site specificity of the inactivation was proved by using the competitive inhibitor p-nitrophenyl-1-thio- β-D-glucopyranoside. pH Dependence studies of inactivation revealed that modification by N-bromoacetyl-β-D-glucopyranosylamine could be directed toward the carboxylate group acting as the catalytic nucleophile, as in the case of the carbodiimide and Woodward reagent K modifications.

Original languageEnglish
Pages (from-to)337-343
Number of pages7
JournalWorld Journal of Microbiology and Biotechnology
Volume21
Issue number3
DOIs
Publication statusPublished - Apr 1 2005

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Keywords

  • Affinity label
  • Aspergillus carbonarius
  • Chemical modification
  • N-bromoacetyl-β-D-glucopyranosylamine
  • β-D-glucosidase

ASJC Scopus subject areas

  • Biotechnology
  • Physiology
  • Applied Microbiology and Biotechnology

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