REACTION OF BETA-D-GLUCOSIDASE A3 FROM ASPERGILLUS-WENTII WITH D-GLUCAL

d‐Glucal acts as mixed competitive/non‐competitive inhibitor against β‐d‐glucosidase A3 from Aspergillus wentii with slow approach to the steady‐state rate of substrate hydrolysis. The same rate is reached whether d‐glucal competes directly with the substrate or the substrate displaces d‐glucal that has been bound by preincubation in the absence of substrate. The Ki for competitive inhibition and the rate constants for the approach to the steady state are in agreement with the kinetic constants for the hydration of d‐glucal to 2‐deoxy‐d‐glucose. The concentration dependence of the inhibition shows that one molecule of d‐glucal (I) binds with complete inhibition but the biphasic dissociation kinetics of the enzyme‐glucal complex and labeling studies point to an additional binding site. An EI2 complex can be isolated at pH 6 and 0°C by rapid ion‐exchange chromatography. This complex can be reactivated at pH 4 and room temperature; all the d‐glucal is released as 2‐deoxy‐d‐glucose. After denaturation of the EI2 complex one molecule of d‐glucal remains bound to the enzyme. The binding site was identified (by isolation and structure determination of a radioactive peptide) as the same aspartate residue that had been labeled with the active‐site‐directed inhibitor, con‐duritol B epoxide, in a previous study. Copyright © 1979, Wiley Blackwell. All rights reserved