Multiple sugar binding sites in alpha-glucosidase
2003
Yao, X. | Mauldin, R. | Byers, L.
Twenty-five analogs of D-glucose were examined as reversible inhibitors of yeast alpha-glucosidase (EC 3.2.1.20). The Ki 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 degrees). All the monosaccharides and the three disaccharides (maltose, isomaltose and alpha,alpha-trehalose) were found to be linear competitive inhibitors with respect to alpha-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 [Ki=1.8(+/- 0.1) mM], one by D-galactose [Ki=164(+/- 11) mM] and one by D-mannose [Ki=120(+/- 9) mM]. The pH dependence for glucose binding closely follows that of V/K [pKa1=5.55(+/- 0.15), pKa2=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.
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