Mechanism of imidazole inhibition of a GH1 β-glucosidase.

Imidazole is largely employed in recombinant protein purification, including GH1 β-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 β-glucosidase from Spodoptera frugiperda (Sfβgly). We confirmed this interaction by showing that imidazole reduces the activity of Sfβgly, 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 Sfβgly 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 β-glucoside. Finally, imidazole interaction in the active site was also demonstrated by showing that it hinders access of carbodiimide to the Sfβgly catalytic residues, protecting them from chemical inactivation. In conclusion, imidazole binds in the Sfβgly active site, generating a partial competitive inhibition. Considering that GH1 β-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.