Resumo

β-Glucosidases activated by glucose and xylose may enhance cellulose saccharification efficiency, being interesting for bioethanol production. The molecular mechanisms of such activation are unknown, and this study aimed the development of kinetic models for the stimulation of a β-glucosidase from Humicola insolens by glucose and xylose. Enzymatic activity was assayed in 50 mmol.L^-1 Bis–Tris buffer, pH 6.0, using p-nitrophenyl-β-D-glucopyranoside as substrate. All proposed kinetic models were analyzed using Matlab software. Intrinsic tryptophan fluorescence emission spectra were measured from 300-450 nm (excitation at 295 nm), and far ultraviolet circular dichroism spectra from 190–250 nm. Biophysical and kinetic data indicated that the enzyme, a monomer of 54 kDa, possesses a catalytic site (CS) and a modulator binding site (MS). Glucose or xylose binding to MS induces conformational changes that stimulate the catalytic activity at CS. Glucose and xylose may compete with the substrate for CS while the substrate competes with the monosaccharides for binding to MS. Stimulation by glucose and xylose is not synergistic and allosteric interactions between MS and CS result in fine modulation of the catalytic activity by the monosaccharides. Kinetic models proposed for glucose or xylose activation adequately described the phenomenon and the simulated values fit the experimental values well. The model developed for enzyme stimulation by glucose in the presence of xylose (and vice versa) showed that a quaternary complex (substrate, xylose and glucose bound to the enzyme) cannot be formed, but two monosaccharide molecules may bind to the enzyme simultaneously, the second one with much lower affinity, possibly at CS. A simplified kinetic model led to a velocity equation that allows prediction of the activity of H. insolens β-glucosidase for known concentrations of substrate, glucose and xylose in the reaction mixture, and thus may be of practical application in cellulose hydrolysis processes.