Enzymic Properties of Purified Myrosinase from Lepidium sativum Seedlings

To establish the substrate specificity o f the thioglucoside glucohydrolase myrosinase (EC 3.2.3.1), this enzyme was purified to homogeneity from light-grown cress (Lepidium sativum L.) seedlings by Sephadex gel filtration. Red Dye and anion exchange (FPLC M ono Q) chro­ matography, and preparative isoelectric focusing. Hydrolytic activity was shown toward only 4 o f the 29 synthetic and natural Oand S-glycosides tested. Highest activity was displayed with the endogenous glucosinolates benzylglucosinolate (ATm, 295 |iM) and sinigrin (Kml 300 |iM) at an optimum pH o f 5.5 in sodium citrate buffer. The synthetic glycosides PNPG (K m, 2.0 m M ) and ONPG were poorer substrates at an optimum pH o f 6.5 in potassium phos­ phate buffer. The enzyme was inactive with all other nitrophenyl glycosides tested including PNP-a-D-glucoside and PNP-thio-ß-D-glucoside, suggesting a requirement for O-ß-D-glucose as the glycone moiety within these substrates. PNPG hydrolysis was stimulated 2.6-fold by ascorbate (1 m M ) . The enzyme exhibited no metal ion requirement and was strongly inhibited by lead nitrate, mercury chloride, and ferric chloride at 1 m M concentration. The metal chela­ tors DIECA, EDTA, o-phenanthroline, and 2,2'-dipyridyl were not inhibitory, but the thiol reagents PCMS, PCMB, and N-ethylmaleimide (at 1 m M ) caused 50-80% inhibition o f enzyme activity.