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

(-)-1-Azafagomine [(3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydroyyridaz ine; inhibitor 1] is a potent glycosidase inhibitor designed to mimic the t ransition state of a substrate undergoing glycoside cleavage. The inhibitio n of glycosidases by inhbitor 1 and analogues has been found to be a relati vely slow process. This 'slow inhibition' process was investigated in the i nhibition of almond beta-glucosidase and yeast isomaltase by inhibitor 1 an d 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 numb er of hydrazine-containing analogues of inhibitor 1 also inhibited beta-glu cosidase and isomaltase slowly, while the amine isofagomine [(3R,4R,5R)-3,4 -dihydroxy-5-hydroxymethylpiperidine; inhibitor 5] only inhibited beta-gluc osidase slowly. Inhibitor 1 and related inhibitors were found to leave almo nd beta-glucosidase with almost identical rate constants, so that the diffe rence 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 i somaltase by inhibitor 1 and a related inhibitor. The values of the rate co nstants were obtained at 25 degrees C and at pH 6.8.