Studies on almond emulsin beta-D-glucosidase. II. Kinetic evidence for independent glucosidase and galactosidase sites.

A purified beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isozyme isolated from almond emulsin was found to catalyze hydrolysis of beta-D-glucopyranosides and beta-D-galactopyranosides but not the corresponding alpha-D-derivatives. Hydrolysis of the corresponding beta-D-thioglycopyranosides at rates 10(3)--10(4) times lower than those for the hydrolysis of the beta-D-glycopyranosides was also noted. The enzyme does not exhibit any transferolytic activity using D-glucose or D-galactose as acceptors. D-glucose, p-nitrothiophenyl-beta-D-glucopyranoside, 5-deoxy-5-thio-D-glucose and D-glucono-delta-lactone are shown to exert mainly competitive inhibition on beta-D-galactopyranoside hydrolysis. D-galactose, p-nitrothiophenyl-beta-D-galactopyranside and methylthio-beta-D-galactopyranoside are shown to inhibit the glucopyranoside hydrolysis mainly non-competitively and to exert competitive inhibition of galactopyranoside hydrolysis. The inhibition caused by the antibiotic Nojirimycin (5-amino-5-deoxy-D-glucose) is shown to be more complex. Analysis of the kinetic data indicates that the catalytic site of the enzyme responsible for the beta-D-glucosidase activity is kinetically distinct from the beta-D-galactosidase site.