Multiple sugar binding sites in α-glucosidase

Abstract

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K i values range from 0.38 mM for 6-deoxy- d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α- p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [ K i=1.8(±0.1) mM], one by d-galactose [ K i=164(±11) mM] and one by d-mannose [ K i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/ K [p K a1=5.55(±0.15), p K a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.