Enzymatic properties of α-d-galactosidase from Trichoderma reesei

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 KI 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 KI 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.