Reversible inhibitors of .beta.-glucosidase

Abstract

A variety of reversible inhibitors of sweet almond .beta.-glucosidase were examined. These included simple sugars and sugar derivatives, amines and phenols. With respect to the sugar inhibitors and, indeed, the various glycoside substrates, the enzyme has what can be considered a "relaxed specificity". No single substituent on glucose, for example, is essential for binding. Replacement of a hydroxyl group with an anionic substituent reduces the affinity while substitution with a cationic (amine) substituent enhances the affinity. Amines, in general, are good inhibitors, binding more tightly than the corresponding alcohols: pKiRNH3+ = 0.645pKiROH + 1.77 (n = 9, r = 0.97). The affinity of a series of 10 primary amines was strongly influenced by substituent hydrophobicity: PKi = 0.52.pi. + 1.32 (r = 0.95). The major binding determinant of the glycoside substrates is the aglycon moiety. Thus, the Ki values of phenols are similar in magnitude to the Ks values of the corresponding aryl .beta.-glucoside. The pH dependence for the inhibition by various phenols indicates that it is the un-ionized phenol which binds to the enzyme when an enzymic group of pKa - 6.8 (.+-.0.1) is protonated. The affinity of the phenol inhibitor is dependent on its basicity with a Bronsted coefficient for binding of .beta. = 0.26 (n = 14, r = 0.98) The pH dependence of the binding of 2 particularly potent .beta.-glucosidase inhibitors was also examined. 1-Deoxynojirimycin (1,5-dideoxy-1,5-imino-D-glucitol) has a pH-corrected Ki = 6.5 .mu.M, and D-glucono-1,5-lactam has a pH-corrected Ki = 29 .mu.M. Although these are 2 of the most effective reversible inhibitors of the enzyme, they are not transition-state analogs. The evidence for this is that their affinities for .beta.-glucosidase do not change with changing pH in a way that completely resembles the influence of pH on kcat/Km for normal substrates as might be expected for an ideal transition-state analog inhibitor.