title: Equilibrium and kinetic studies on the binding of gluconolactone to almond beta-glucosidase in the absence and presence of glucose.
abstract: The binding of glucono-1,5-lactone (gluconolactone) with almond beta-glucosidase was studied at pH 5.0 and 25 degrees C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, delta Fmax, were determined to be 12.7 microM and 14.7%, respectively. From the study of the temperature dependence of Kd, delta G degrees, delta H degrees and delta S degrees for the binding were evaluated to be -6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25 degrees C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism: (formula; see text) which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k-1/k+1 = 4.5 mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2 = 94 s-1, k-2 = 0.36 s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D005942:Gluconates / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D005959:Glucosidases / Q000378:metabolism*; D007700:Kinetics; D007783:Lactones; D010944:Plants / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000378:metabolism*
chemical_list: D005942:Gluconates; D007783:Lactones; D005959:Glucosidases; D001617:beta-Glucosidase; D005947:Glucose; C010730:beta-glucono-1,5-lactone