Subsite structure of the beta-glucosidase from Aspergillus niger, evaluated by steady-state kinetics with cello-oligosaccharides as substrates.

The beta-glucosidase from a commercially available preparation from Aspergillus niger was highly purified. The Michaelis constant Km and the molar activity K0 for cello-oligosaccharide substrates Gn (n = 2-6) were obtained by steady-state kinetic analysis on the beta-glucosidase-catalyzed hydrolysis at 25 degrees C and pH 5.0. Stoichiometric production of Gn-1 by the beta-glucosidase reaction for Gn was confirmed by HPLC techniques. Based on Km and K0 for Gn, subsite affinities (Ai, i = 1-6) were estimated as follows (kcal/mol): A1 = 1.3, A2 = 5.2, A3 = 0.65, A4 = -0.10, A5 = -0.65, and A6 = -0.26, of which A1-A3 are much higher than those of the beta-glucosidase of Candida wickerhamii. The subsite structure is quite similar to that of the alpha-glucosidase of A. niger, whereas the dependence of k0 on n is highly characteristic for beta-glucosidase, and decreases with n, suggesting some interaction between the particular subsites.