High resolution x-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base

Myrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio beta -D-gluco sides (glucosinolates) considered part of the plant defense system. Althoug h O-glycosidases are ubiquitous, myrosinase is the only known S-glycosidase . Its active site is very similar to that of retaining O-glycosidases, but one of the catalytic residues in O-glycosidases, a carboxylate residue func tioning as the general base, is replaced by a glutamine residue. Myrosinase is strongly activated by ascorbic acid. Several binary and ternary complex es of myrosinase with different transition state analogues and ascorbic aci d have been analyzed at high resolution by x-ray crystallography along with a 2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors, D- gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a mimic of the enzyme-substrate complex, Ascorbate binds to a site distinc t from the glucose binding site but overlapping with the aglycon binding si te, suggesting that activation occurs at the second step of catalysis, i.e. hydrolysis of the glycosyl enzyme, A water molecule is placed perfectly fo r activation by ascorbate and for nucleophilic attack on the covalently tra pped S-fluoroglucosyl-moiety, Activation of the hydrolysis of the glucosyl enzyme intermediate is further evidenced by the observation that ascorbate enhances the rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion that ascorbic acid substitutes for the catalytic base in myrosinase.