The effect of glycerol on the activity of β-glucosidase from Botryodiplodia theobromae Pat

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Abstract
In the activity of the high-Mr.beta.-glucosidase A (.beta.-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. on o-nitrophenyl .beta.-D-glucopyranoside as substrate, both Vmax and Km increased non-linearly with increasing concentration of glycerol, and the Vmax/Km(app.) ratio decreased non-linearly with increasing concentration of glycerol. 2. No increase in rate was observed with phenyl .beta.-D-glucopyranoside as substrate in the presence of up to 250 mM-glycerol, indicating that glucosylation is rate-limiting with this substrate. 3. With o-nitrophenyl .beta.-D-glucopyranoside, p-nitrophenyl .beta.-D-glucopyranoside and phenyl .beta.-D-glucopyranoside and phenyl .beta.-D-glucopyranoside as substrates, kcat. values of 793.7 s-1, 62.8 s-1 and 5.4 s-1 respectively were calculated. 4. With o-nitrophenyl .beta.-D-glucopyranoside and phenyl .beta.-D-glucopyranoside as substrate, .alpha.-deuterium kinetic isotope effects of 1.9 .+-. 0.03 and 1.01 .+-. 0.01 respectively were found; in the presence of 200 mM-glycerol the values were 1.21 .+-. 0.03 and 1.02 .+-. 0.01 respectively. 5. In the presence of a large excess of o-nitrophenyl .beta.-D-glucopyranoside ([S] = 35.7 Km), the amount of o-nitrophenol and also of the transglucosylation product formed by .beta.-glucosidase action increased nonlinearly, whereas that of glucose formed decreased non-linearly with increasing glycerol concentration. 6. All these results were found to fit the data calculated from rate equations derived on the basis of the proposed mechanism of enzyme action involving two ion-pair intermediates and a covalent .alpha.-D-glucosyl-enzyme in the reaction sequence [Umezurike (1987) Biochem. J. 241, 455-462].