INVESTIGATION OF THE ACTIVE-SITE OF THE CYANOGENIC BETA-D-GLUCOSIDASE(LINAMARASE) FROM MANIHOT-ESCULENTA CRANTZ (CASSAVA) .1. EVIDENCE FORAN ESSENTIAL CARBOXYLATE AND A REACTIVE HISTIDINE RESIDUE IN A SINGLECATALYTIC CENTER

The broad-specificity cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crant z (cassava) was kinetically characterized in mixed substrate systems a nd with the transition-state analogue glucono(1-5)lactone and a series of 1-thio substrate analogues. The results indicate a common catalyti c and a common sugar binding site in the enzyme for all of the investi gated substrates. Kinetic parameters of the hydrolysis of linamarin an d p-nitrophenyl beta-D-glucopyranoside were determined over the pH ran ge 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 lina marin or p-nitrophenyl beta-D-glucopyranoside as the substrate. Kineti c analysis of the modification indicated that one molecule of water-so luble 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 beta-D-glucopyranosi de, beta-D-glucopyranosylamine, and glucono(1-5)lactone. Spectrophotom etric analysis at 340 nm showed that from the three carboxylate groups modified by Woodward's reagent K essentially one was protected by p-n itrothiophenyl beta-D-glucopyranoside. During modification V-max decre ased to 30% of that of the unmodified enzyme and K-m remained unchange d. The pH dependence of inactivation showed the involvement of a group with a pK value of 4.6, indicating the modification of a carboxyl res idue essential for activity. Treatment of the enzyme with the histidin e-group-specific reagent diethylpyrocarbonate resulted in 80% loss of enzyme activity, in biphasic kinetics. A treatment with 0.5 M hydroxyl amine at pH 7.0 regenerated 92% of the original enzyme activity. The p resence of the competitive inhibitor beta-D-glucopyranosylamine protec ted the enzyme against inactivation, preventing the modification of on e histidine residue. Statistical analysis of the residual fractional a ctivity against the number of modified residues indicated that the mod ification of one histidine is responsible for 40-50% of the inactivati on. The pH dependence of the inactivation gave a pK value of 7.0 for t he histidine group upon which the activity depends. During modificatio n, V-max decreased to 30% and K-m decreased to 50% of the original val ues. (C) 1994 Academic Press, Inc.