Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside by cellobiase of Trichoderma viride

Abstract

Cellobiase has been isolated from the crude cellulase mixture of enzymes of Trichoderma viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose have been investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature, and dielectric constant, using isopropanol-buffer mixtures. The results show that (i) there is a marked activation of the reaction by initial glucose concentrations of 4 X 10(-3) M to 9 X 10(-2) M and strong inhibition of the reaction at higher initial concentrations, (ii) the log rate -pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, (iii) the energy of activation at pH 5.1 is 10.2 kcal mol-1 over the temperature range 5-56 degrees C, and (iv) the rate decreases from 0 to 20% (v/v) isopropanol. The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-beta-D-glucoside was examined by pre-steady-state methods in which [enzyme]0 greater than [substrate]0, and by steady-state methods as a function of pH and temperature. The results show (i) a value for k2 of 21 S-1 at pH 7.0 (where k2 is the rate constant for the second step in the assumed two-intermediate mechanism (formula: see text), (ii) a log rate -pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5-6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3), and (iii) an activation energy of 12.5 kcal mol-1 at pH 5.7 over the temperature range 10-60 degrees C.