An unusual case of ‘uncompetitive activation’ by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings

Abstract

Myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) is a plant enzyme that hydrolyses glucosinolates, principally to isothiocyanates. Myrosinase was purified to homogeneity in good yield from 8-day-old seedlings of Raphanus sativus (daikon) using a four-step procedure involving chromatographies on anion exchange, hydrophobic Phenyl-Sepharose, gel filtration and concanavalin A-Sepharose. In order to stabilize the enzyme and to avoid excessive peak broadening during chromatography, 30% (v/v) glycerol was added to dialysis and chromatography buffers. The purified enzyme was eluted as a single peak from a gel-filtration sizing column with an apparent molecular mass of 120 kDa. The enzyme was resolved into two subunits with molecular masses of 61 and 62 kDa by SDS/PAGE. Ascorbic acid activated the purified enzyme more than 100-fold. The Vmax and Km values for the hydrolysis of allyl glucosinolate (sinigrin) were 2.06 μmol/min per mg of protein and 23 μM in the absence of ascorbate and 280 μmol/min per mg of protein and 250 μM in the presence of 500 μM ascorbate, respectively. As the ascorbate concentration was increased from 50 to 500 μM, the Vmax and Km values increased in parallel, and thus the Vmax/Km ratio remained constant. Similarly, raising the concentrations of sinigrin increased the concentration of ascorbic acid required for half-maximal activation (Ka). At a sinigrin concentration of 250 μM, the Ka for ascorbic acid was 55 μM. Sulphate, a reaction product, was a competitive inhibitor of activity, having a Ki of 60 mM with respect to sinigrin and of 27 mM with respect to ascorbate. Thus activation of myrosinase from R.sativus by ascorbic acid exemplifies an unusual and possibly unique example of linear ‘uncompetitive activation’ (i.e. a proportionate increase in Vmax and Km) of an enzyme. The enzyme also had β-glucosidase activity and hydrolysed p-nitrophenyl-β-D-glucopyranoside.