Characterization of high pI α-glucosidase from germinated barley seeds: substrate specificity, subsite affinities and active-site residues

Abstract

Substrate specificity and subsite affinities of high pl α-glucosidase from germinated barley ( Hordeum vulgare L.) seeds were investigated by kinetics. The enzyme has only one maltose binding site per molecule and shows high activity on small maltooligosaccharides and nigerose. Hydrolysis of isomaltose and p-nitrophenyl α-glucoside is moderate. Trehalose is not hydrolyzed at detectable rates. The ratios of the maximum velocities for maltose, nigerose, isomaltose, p-nitrophenyl α-glucoside and malto-triose, -tetraose, -pentaose, -hexaose, -heptaose are 100:95:21:9:111:116:119:104:111. The K m values for these substrates are 1.91, 1.29, 5.32, 1.04, 1.11, 2.37, 2.92, 5.44 and 7.89 mM, respectively. Based on the rate parameters for maltooligosaccharides, the subsite affinities ( A i s) in the active site of the enzyme were evaluated according to subsite theory. Subsites 1, 2 and 3, having positive A i values ( A 1, A 2 and A 3; 1.34, 5.37 and 0.27 kcal/mol, respectively), were considered to be effective for the binding of substrate to the active site. The different arrangement of subsite affinities among α-glucosidases, glucoamylases and amylases was used to explain their substrate specificities.