CHEMICAL MODIFICATION OF BETA-GLUCOSIDASE FROM TRICHODERMA-REESEI QM-9414

The inhibition of beta-glucosidase from Trichoderma reesei QM 9414 by several specific reagents was studied. Diethylpyrocarbonate (DEP) near ly abolished the enzyme activity at concentrations above 10 mM. The pr esence of substrate or analogs protected the enzyme against inactivati on. The reaction followed pseudo-first order kinetics with a second-or der rate constant of 0.02 mM(-1).min(-1). The pH-dependence of the ina ctivation showed the involvement of a group with a pK of 5.2. Differen ce spectra at 242 nm and the reversal of the inactivation in the prese nse of 1 M hydroxylamine indicated the modification of histidine resid ues. Statistical analysis of residual fractional activity versus the n umber of modified histidine residues indicated that one histidine resi due is essential for catalysis. p-Hydroxymercuribenzoate completely in hibited the enzyme at concentrations of the reagent above 2 mM. Substr ate or analogs protected the enzyme against inactivation. The reaction followed pseudo-first order kinetics with a second-order rate constan t of 0.002 mM(-1). min(-1). Treatment of the modified enzyme with 5,5' -dithio-bis(2-nitrobenzoic acid) (DTNB) showed that one cysteine resid ue was essential for activity. At pH 5.0 2-ethoxy-1-ethoxy-carbonyl-1, 2-dihydroquinoline (EEDQ) inactivated the enzyme according to pseudo-f irst order kinetics with a second-order rate constant of 0.12 min(-1). The pH-dependence of the inactivation showed the involvement of a gro up with a pK of 5.64, indicating the modification of a carboxyl group essential for activity.