RESUMO
Imidazole is largely employed in recombinant protein purification, including GH1 ß-glucosidases, but its effect on the enzyme activity is rarely taken into consideration. Computational docking suggested that imidazole interacts with residues forming the active site of the GH1 ß-glucosidase from Spodoptera frugiperda (Sfßgly). We confirmed this interaction by showing that imidazole reduces the activity of Sfßgly, which does not result from enzyme covalent modification or promotion of transglycosylation reactions. Instead, this inhibition occurs through a partial competitive mechanism. Imidazole binds to the Sfßgly active site, reducing the substrate affinity by about threefold, whereas the rate constant of product formation remains unchanged. The binding of imidazole within the active site was further confirmed by enzyme kinetic experiments in which imidazole and cellobiose competed to inhibit the hydrolysis of p-nitrophenyl ß-glucoside. Finally, imidazole interaction in the active site was also demonstrated by showing that it hinders access of carbodiimide to the Sfßgly catalytic residues, protecting them from chemical inactivation. In conclusion, imidazole binds in the Sfßgly active site, generating a partial competitive inhibition. Considering that GH1 ß-glucosidases share conserved active sites, this inhibition phenomenon is probably widespread among these enzymes, and this should be taken into account when considering the characterization of their recombinant forms.
Assuntos
Glucosídeos , beta-Glucosidase , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Domínio Catalítico , Hidrólise , Imidazóis/farmacologiaRESUMO
In this work, we investigated how activity and oligomeric state are related in a purified GH1 ß-glucosidase from Spodoptera frugiperda (Sfßgly). Gel filtration chromatography coupled to a multiple angle light scattering detector allowed separation of the homodimer and monomer states and determination of the dimer dissociation constant (KD ), which was in the micromolar range. Enzyme kinetic parameters showed that the dimer is on average 2.5-fold more active. Later, we evaluated the kinetics of homodimerization, scanning the changes in the Sfßgly intrinsic fluorescence over time when the dimer dissociates into the monomer after a large dilution. We described how the rate constant of monomerization (koff ) is affected by temperature, revealing the enthalpic and entropic contributions to the process. We also evaluated how the rate constant (kobs ) by which equilibrium is reached after dimer dilution behaves when varying the initial Sfßgly concentration. These data indicated that Sfßgly dimerizes through the conformational selection mechanism, in which the monomer undergoes a conformational exchange and then binds to a similar monomer, forming a more active homodimer. Finally, we noted that conformational selection reports and experiments usually rely on a ligand whose concentration is in excess, but for homodimerization, this approach does not hold. Hence, since our approach overcomes this limitation, this study not only is a new contribution to the comprehension of GH1 ß-glucosidases, but it can also help to elucidate protein interaction pathways.