The mechanism of action of β-glucosidase from Botryodiplodia theobromae Pat

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Abstract
1. The activity of the high-molecular-weight .beta.-glycosidase (.beta.-D-glucoside glucohydrolase, EC3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. was affected by added NaCl in such a way that an initial phase of stimulation was followed by a phase of rapid non-linear decrease in velocity and finally by a phase of slow decrease in velocity as the concentration of NaCl was increased. 2. In the presence of 0.014 M-sodium acetate/acetic acid buffer (pH 5.0) there was a slight increase in enzymic activity in the presence of low concentrations of dioxan (up to about 10% dioxan) and a rapid decrease in enzymic activity at higher dioxan concentrations, but both effects were mitigated in the presence of 0.1 M buffer. 3. The order of efficiency of added glucosyl acceptors in .beta.-glucosidase-catalysed reactions was found to be fructose > sucrose > glycerol > methanol. 4. The enzyme was inactivated by the active-site-directed compound conduritol-B-epoxide; but this inactivation was concentration-dependent, was prevented by 10 mM-glucose, and involved an acidic group with pKa 4.3. 5. A rate equation has been derived on the assumption of a mechanism of action involving a solvent-separated an an intimate glucosyl cation-carboxylate ion-pair intermediate and an .alpha.-glucosyl enzyme intermediate [Umezurike, G.M. (1981) Biochem. J. 199, 203-209]. 6. Calculations based on the application of the derived rate equation and the calculated kinetic parameters show that the rate equation explains the peculiar properties of .beta.-glucosidase in the presence of added glucosyl acceptors or of NaCl.