Investigation of the slow inhibition of almond β-glucosidase and yeast isomaltase by 1-azasugar inhibitors: Evidence for the 'direct binding' model

Abstract

(-)-1-Azafagomine [(3R,4R,5R)-4,5-dihydroxy-3-hydroxy-methylhexahydropyridazine; inhibitor 1] is a potent glycosidase inhibitor designed to mimic the transition state of a substrate undergoing glycoside cleavage. The inhibition of glycosidases by inhbitor 1 and analogues has been found to be a relatively slow process. This 'slow inhibition' process was investigated in the inhibition of almond β-glucosidase and yeast isomaltase by inhibitor 1 and analogues. Progress-curve experiments established that the time-dependent inhibition of both enzymes by inhibitor 1 was a consequence of relatively slow dissociation and association of the inhibitor from and to the enzyme, and not a result of slow interchanges between protein conformations. A number of hydrazine-containing analogues of inhibitor 1 also inhibited β-glucosidase and isomaltase slowly, while the amine isofagomine [(3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine; inhibitor 5] only inhibited β-glucosidase slowly. Inhibitor 1 and related inhibitors were found to leave almond β-glucosidase with almost identical rate constants, so that the difference in K(i) values depended almost entirely on changes in the binding rate constant, k(on) . The same trend was observed for the inhibition of yeast isomaltase by inhibitor 1 and a related inhibitor. The values of the rate constants were obtained at 25°C and at pH 6.8.