Investigation of the active site of the cyanogenic β-D-Glucosidase (linamarase) from manihot esculenta crantz (cassava). I. evidence for an essential carboxylate and a reactive histidine residue in a single catalytic center

Zsolt Keresztessy, László Kiss, Monica A. Hughes

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The broad-specificity cyanogenic β-D-glucosidase (β-D-glucoside glucohydrolase, EC (linamarase) from Manihot esculenta Crantz (cassava) was kinetically characterized in mixed substrate systems and with the transition-state analogue glucono(1-5)lactone and a series of 1-thio substrate analogues. The results indicate a common catalytic and a common sugar binding site in the enzyme for all of the investigated substrates. Kinetic parameters of the hydrolysis of linamarin and p-nitrophenyl β-D-glucopyranoside were determined over the pH range 3.5-9.0. The pH-dependence curves gave apparent pK values of 4.5 (4.6) and 7.1 (7.3) for the free enzyme, while values of 4.1 (3.7) and 9.3 were obtained for the enzyme-substrate complexes, using either linamarin or p-nitrophenyl β-D-glucopyranoside as the substrate. Kinetic analysis of the modification indicated that one molecule of water-soluble carbodiimide or Woodward′s reagent K is required to bind to the enzyme for inactivation. The enzyme was protected against inactivation by the competitive inhibitors p-nitrothhiophenyl β-D-glucopyranoside, β-D-glucopyranosylamine, and glucono(1-5)lactone. Spectrophotometric analysis at 340 nm showed that from the three carboxylate groups modified by Woodward′s reagent K essentially one was protected by p-nitrothiophenyl β-D-glucopyranoside. During modification Vmax decreased to 30% of that of the unmodified enzyme and Km remained unchanged. The pH dependence of inactivation showed the involvement of a group with a pK value of 4.6, indicating the modification of a carboxyl residue essential for activity. Treatment of the enzyme with the histidine-group-specific reagent diethylpyrocarbonate resulted in 80% loss of enzyme activity, in biphasic kinetics. A treatment with 0.5 M hydroxylamine at pH 7.0 regenerated 92% of the original enzyme activity. The presence of the competitive inhibitor β-D-glucopyranosylamine protected the enzyme against inactivation, preventing the modification of one histidine residue. Statistical analysis of the residual fractional activity against the number of modified residues indicated that the modification of one histidine is responsible for 40-50% of the inactivation. The pH dependence of the inactivation gave a pK value of 7.0 for the histidine group upon which the activity depends. During modification, Vmax decreased to 30% and Km decreased to 50% of the original values.

Original languageEnglish
Pages (from-to)142-152
Number of pages11
JournalArchives of Biochemistry and Biophysics
Issue number1
Publication statusPublished - Oct 1994



  • Cassava
  • Chemical modification
  • Enzyme inhibition
  • Glucono(l-5)lactone
  • Thioglycosides
  • β-glucosidase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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