ENERGY OF BINDING OF ASPERGILLUS-ORYZAE BETA-GLUCOSIDASE WITH THE SUBSTRATE, AND THE MECHANISM OF ITS ENZYMIC ACTION

  • 1 January 1983
    • journal article
    • research article
    • Vol. 94  (5) , 1637-1647
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 A. 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 Km of phenyl .beta.-glucoside and its deoxy analogs, 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 [phenyl .beta.-D-glucopyranoside] 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 [phenyl .beta.-D-xylopyranoside]. 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 distorted to a half chair form which is labile to acid hydrolysis. Kinetic data, especially kcat, showed that hydroxyls of C-2, C-3 and C-4 played important roles not only in the binding of the substrate to the active site, but also in the increase of the hydrolysis rate.