Enzymatic properties of α-D-galactosidase from Trichoderma reesei

Article abstract of DOI:10.1016/S0008-6215(96)00248-0

The kinetics of hydrolysis of a number of natural and synthetic substrates [melibiose, raffinose, stachyose, methyl α-D-galactopyranoside, and p-nitrophenyl α-D-galactopyranoside (PNPG)], catalyzed by α-D-galactosidase from the fungus Trichoderma reesei, has been studied. A number of N-acyl-α-D-galactopyranosylamines, which are competitive inhibitors of α-D-galactosidase, have been synthesized, and the K(I) values for these compounds have been obtained. The inhibiting properties of the competitive inhibitors of D-galactose, 1,5-anhydro-D-galactitol, and 2-deoxygalactose have been compared, and reasons for differences in K(I) values between these compounds have been discussed. It has been shown that α-D-galactosidase exhibits transglycosylating activity; the main product of transglycosylation in the reaction with PNPG is p-nitrophenyl 6-O-α-D-galactopyranosyl-α-D-galactopyranoside. The hydrolysis inhibition in the presence of a substrate has been shown to correlate with the substrate transglycosylation. Data of steady-state kinetics together with data of presteady-state kinetics obtained by the stop-flow method suggest that an intermediate galactosyl-enzyme complex is formed in the reaction and is of particular importance in the processes under study. A minimal kinetic scheme describing the experimental data obtained is proposed.