The ,B-glucosidase from Botryodiplodia theobromae

1. In the presence of a high concentration ofp-nitrophenyl JJ-D-glucopyranoside (donor) the rates of production of p-nitrophenol and a transglucosylation product (1-glyceryl f,-D-glucopyranoside) increased, whereas the rate of production of glucose decreased with increasing concentration of glycerol in reactions catalysed by the.high-molecular-weight ,-glucosidase (fi-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. 2. When [donor] >,Km the rate of production of p-nitrophenol was higher in the presence of glycerol than in its absence, whereas when [donor] <Km the rate of production of p-nitrophenol was lo'wer in the presence of glycerol than in its absence. 3. Glycerol' increased both the Michaelis constant (Ki) and maximum velocity (Vmax,), whereas dioxan increased Km but decreased Vmax. 4. Up to 1 mM-AgNO3 had no effect on enzyme activity. 5. A 2H-solvent-isotope-effect [VmU.(H2O)/Vm,.(2H2O)] value of 1.40 + 0.05 was found at pH (or p2H) 5.8. 6. a-2H-kinetic-isotope-effect (kH/k2H) values of 1.03+0.01 and 1.05 + 0.01 were found in the absence and presence of glycerol respectively. 7. Although maltose was a non-competitive inhibitor of fJ-glucosidase activity, the ratio of velocity in the presence of glycerol to that in its absence increased, after an initial decline, with increasing concentration of maltose. 8. These results are discussed in terms of a mechanism involving a solvent-separated glucosyl cation-carboxylate ion-pair, which has greater affinity for alcoholic glucosyl acceptors, and an intimate ion-pair, which has greater affinity for water as a glucosyl acceptor and which could collapse reversibly and rapidly into a preponderance of an unreactive covalent glucosyl-enzyme.