ABSTRACT
The induced beta-D-glucosidase from Stachybotrys atra hydrolyzes aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by the same basic two-step mechanism. In the first step the aglycon group is split of with simultaneous formation of an enzyme-glycosyl complex. In the second step this intermediate complex reacts with water yeilding beta-D-glucose or beta-D-xylose. For beta-D-xyloside hydrolysis each of the two steps is partially rate-controlling, whereas for beta-D-glucoside hydrolysis the second step is rate-limiting. The enzyme is inhibited by high concentrations of substrate and the exact rate-concentration equation is a second-order equation. 1-Thio-beta-D-glycopyranosides with an aromatic aglycon inhibit the reaction in both a competitive and non-competitive way. A tentative mechanism is proposed to explain all types of inhibition. In this mechanism substrates and inhibitors with an aromatic aglycon group bind through hydrophobic forces to the 'aglycon subsite' of the intermediate enzyme-glycosyl complex. Binding of the second substrate molecule or of the inhibitor to this complex does not prevent the reaction of the glycosyl moiety with water, it only decreases the rate of the second step.
Subject(s)
Glucosidases/biosynthesis , Glucosides/metabolism , Glycosides/metabolism , Mitosporic Fungi/enzymology , Stachybotrys/enzymology , Xylose/analogs & derivatives , beta-Glucosidase/biosynthesis , Enzyme Induction , Galactosides/pharmacology , Kinetics , Substrate Specificity , Thioglycosides/pharmacology , Xylose/metabolism , beta-Glucosidase/antagonists & inhibitorsABSTRACT
We have purified an induced beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Stachybotrys atra to apparent homogeneity. The induced enzyme was clearly different from the constitutive beta-D-glucosidase. The molecular weight was 65 500-69 000, the pH optimum was at 6.7 and the isoelectric point at 4.8. Carbohydrate content (related to glucose) was 14.4%. The enzyme showed beta-D-glucosidase, beta-D-xylosidase and beta-D-thioglucosidase activity. These three activities sppear to be due to the same enzyme. The enzyme was strongly inhibited by D-glucono-(1 leads to 5)-lactone and nojirimycin and was very sensitive to sulfhydryl reagents.
