Mechanism-based inhibition and stereochemistry of glucosinolate hydrolysis by myrosinase.

Myrosinase is a particular glucosidase which hydrolyzes a variety of plant 1-thio-beta-D-glucosides known as the glucosinolates. This enzyme, which is the only glycosidase able to hydrolyze these naturally occurring thioglucosides, has been found previously to display strong sequence similarities with family 1 O-glycosidases. Myrosinase therefore offers the opportunity to compare the mechanism of enzymatic cleavage of S- vs O-glycosidic bonds. The stereochemistry of hydrolysis of sinigrin by Sinapis alba myrosinase was followed by 1H NMR and the enzyme was found to operate with a mechanism retaining the anomeric configuration at the cleavage point exactly like the related O-glycosidases found in family 1. Myrosinase was readily inactivated by 2-deoxy-2-fluoroglucotropaeolin with inactivation kinetic parameters of Ki = 0.9 mM and ki = 0.083 min-1. Reactivation kinetic parameters were determined in buffer only, with k(react) = 0.015 h-1 and t1/2 = 46 h, and also in the presence of acceptors of transglycosylation. No significant changes were observed in the presence of methyl beta-D-glucoside, but with azide anion the half-life of reactivation was found to be reduced to t1/2 = 20 h. These results suggest that myrosinase inhibition by 2-deoxy-2-fluoroglucotropaeolin occurs via the accumulation of a long-life glucosyl-enzyme intermediate and that the catalytic machinery of the enzyme is composed of only one catalytic residue, a nucleophilic glutamate, while the acid catalyst residue found in the corresponding O-glycosidases is missing.