Multiple sugar binding sites in alpha-glucosidase.
Twenty-five analogs of D-glucose were examined as reversible inhibitors of yeast alpha-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 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 [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 [pK(a1)=5.55(+/-0.15), pK(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.
Yao
Xiaojie
X
Department of Chemistry, Tulane University, 6400 Freret St., New Orleans, LA 70118-5698, USA.
Mauldin
Rebecca
R
Byers
Larry
L
D001665:Binding Sites; D001667:Binding, Competitive; D005690:Galactose / Q000494:pharmacology; D005947:Glucose / Q000031:analogs & derivatives* / Q000494:pharmacology; D005960:Glucosides / Q000378:metabolism*; D065089:Glycoside Hydrolase Inhibitors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008358:Mannose / Q000494:pharmacology; D008958:Models, Molecular; D012441:Saccharomyces cerevisiae; D000520:alpha-Glucosidases / Q000737:chemistry / Q000378:metabolism
D005960:Glucosides; D065089:Glycoside Hydrolase Inhibitors; C025193:4-nitrophenyl beta-D-glucoside; D000520:alpha-Glucosidases; D005947:Glucose; D008358:Mannose; D005690:Galactose