Active-site-directed inactivation of Schizophyllum commune cellulase by 4′, 5′-epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside

Abstract

4′,5′-Epoxypentyl-4-D-(β-D-glucopyranosyl)-β-D-glucopyranoside (4) was synthesized by a Koenigs–Knorr reaction of 4-penten-1-ol and acetobromcellobiose, promoted by silver trifluoromethanesulfonate and N,N′-tetramethylurea, and tested as a potential active-site-directed irreversible inhibitor of the Schizophyllum commune cellulase. Incubation of the S. commune cellulase with 4 resulted in a time-dependent irreversible inactivation of the enzyme. The inactivation process obeyed pseudo-first-order kinetics and the hyperbolic plot of kobs as a function of inhibitor concentration provided values for Kd and k2 of 150 mM and 2.0 × 10−4 s−1, respectively, at pH 5.5 and 25 °C. The binding of a competitive inhibitor, cellobiose, to the cellulase prior to incubation with 4 protected the enzyme from rapid inactivation, suggesting that the inactivation is due to attack at the active site. The dependence of the inactivation on pH is consistent with the participation of carboxyl groups. Treatment of the affinity-labeled enzyme with [14C]methoxyamine resulted in the near stoichiometric formation of a stable radiolabelled adduct, suggesting that one inhibitor molecule binds per enzyme active site of the enzyme.