STUDIES ON THE MECHANISM OF MYROSINASE - INVESTIGATION OF THE EFFECT OF GLYCOSYL ACCEPTORS ON ENZYME-ACTIVITY

Myrosinase (EC 3.2.3.1) is the beta-thioglucosidase enzyme responsible for the hydrolysis of glucosinolates, a group of naturally occurring plant metabolites. The enzyme catalyzes the hydrolysis of these S-gluc osides to give D-glucose and an aglycone fragment, which then rearrang es to give sulfate and an isothiocyanate. As part of ongoing mechanist ic studies on myrosinase, the ability of the enzyme to catalyze transg lycosylation reactions has been examined. Enzyme activity and stabilit y were both decreased in the presence of various organic solvents, inc luding simple alcohols, but not sufficiently to prevent reaction takin g place. However, in contrast to most other beta-glycosidases, myrosin ase did not catalyze transglycosylation reactions either with the alco hols or other suitable glycosyl accepters. Although a wide range of po tential accepters were investigated, none proved to be effective. Even when appropriately charged side chains were included in the acceptor molecule to mimic the sulfonic acid in the glucosinolate structure, tr ansglycosylation did not take place. The putative enzyme-glycosyl inte rmediate therefore appears to be unavailable for reaction, possibly be cause D-glucose is the first product released from the enzyme. The tra nsition state analogue, glucono-delta-lactone, a potent competitive in hibitor of beta-glucosidase, was found to be a poor noncompetitive inh ibitor of myrosinase. Myrosinase is specifically activated by ascorbic acid, and it is proposed that the inhibitor is binding at this altern ative site.