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  • Title: Energy of binding of Aspergillus oryzae beta-glucosidase with the substrate, and the mechanism of its enzymic action.
    Author: Mega T, Matsushima Y.
    Journal: J Biochem; 1983 Nov; 94(5):1637-47. PubMed ID: 6418735.
    Abstract:
    Several beta-D-glucopyranosides (p-nitrophenyl, phenyl, and ethyl), 1-thio-beta-D-glucopyranosides, and phenyl 2-deoxy, 3-deoxy, 4-deoxy, and 6-deoxy beta-D-glucopyranosides were synthesized and used to study the mechanism of the enzymatic action of Taka-beta-glucosidase [EC 3.2.1.21 Aspergillus oryzae]. Kinetic constants of the enzyme for these glycosides were determined from S/V-S or 1/V-1/S plots, and the hydrolysis rates of these compounds with the enzyme, acid (3 N HCl) and alkali (3 N NaOH) were compared. Inhibition of the enzyme by 1,5-anhydroglucitol, glucal, dihydroglucal, and 1,6-anhydroglucopyranose was also examined. Glucal and 1,5-anhydroglucitol showed strong competitive inhibition. Free energy of binding of each hydroxyl group of glucosidic glucose with the enzyme was estimated from Kms of phenyl beta-glucoside and its deoxy analogues, and also Ki values of some inhibitors. The free energies of binding of 2-OH, 3-OH, 4-OH, and 6-OH were calculated to be 1.1, 2.4, 0.7, and 1.8 kcal/mol, respectively. The free energy of binding of phenoxide at C-1 (0.3 kcal/mol) was calculated from the Km of Ph-beta-Glc and Ki of 1,5-anhydroglucitol. The energy of binding of 5-CH2OH (2.3 kcal/mol) was obtained from the Km of Ph-beta-Glc and that of Ph-beta-Xyl. The sum (6.8 kcal/mol) of each partial binding free energy was close to the value of binding free energy of Ph-beta-Glc (7.0 kcal/mol) calculated by the equation; -delta Gbind = -RT ln Km-T delta Smix, showing that the methods of estimation of each binding energy used in the present study seemed reasonable. Glucal, having a pyranose form distorted slightly, showed strong competitive inhibition and the Ki of this inhibitor was smaller than the Km of Ph-beta-Glc, suggesting that the sugar ring bound to the active site was distored to a half chair form which is labile to acid hydrolysis.
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