Inhibition of beta-glucosidases from almonds by cationic and neutral beta-glucosyl derivatives

The beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isoenzymes B from sweet and bitter almonds showed considerable differences in their kinetic and inhibition parameters, but both were inhibited much more strongly by basic beta-glucosyl derivatives than by their neutral analogs. The additional interaction energy apparently due to the basic character ranged from 18 kJ/mol (4.3 kcal/mol) for beta-glucosylamine compared to beta-glucose to 28 kJ/mol (6.9 kcal/mol) for N-benzyl-beta-glucosylamine compared to N-beta-glucosyl-p-toluidine. N-beta-Glucosylpyridinium ion and N-beta-glucosylimidazol which both cannot be protonated at the glucosylated nitrogen are very weak inhibitors. beta-2-Amino-2-deoxyglucose is bound with half the affinity of beta-glucosylamine. The structural requirement for strong inhibition is thus the protonation of the inhibitor at the glucosylated nitrogen. The additional binding energy is assumed to be due to the electrostatic interaction of the inhibitor cation with a carboxylate group in an environment of low polarity. The failure of the pyridinium ion to show this interaction is attributed to the presence of a positively charged group at the active site which acts as proton donor. The pKa values of beta-glucosylamine and its derivatives have been determined and found to be 3.5 units lower than those of the corresponding parent amines. An exception is beta-glucosylimidazol (pKa 5.4) which is protonated on the non-glycosylated nitrogen.