Mechanism of imidazole inhibition of a GH1 beta-glucosidase

Author(s)

Chagas, Rafael S. ; Otsuka, Felipe A. M. ; Pineda, Mario A. R. ; Salinas, Roberto K. ; Marana, Sandro R.

• Document type: Journal article
• Source: FEBS OPEN BIO; v. 13, n. 5, p. 14-pg., 2023-03-25.

Abstract

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

• FAPESP's process: 18/25952-8 - Catalysis and Thermostability in Enzymes: Oligomerization, Structure Network and Dynamics Effects
• Grantee: Sandro Roberto Marana
• Support Opportunities: Regular Research Grants