Di- and Oligosaccharide Substrate Specificities and Subsite Binding Energies of Pig Intestinal Glucoamylase-Maltase

The substrate specificity of pig intestinal glucoamylase-maltase was investigated. The α-1,β-2-glycosidic bond of the disaccharide sucrose was not hydrolyzed. Various substrates with α-1,4-glycosidic bonds (maltose, maltooligosaccharides) were hydrolyzed with high maximal reaction velocities. Reduction lowered the rate of hydrolysis drastically:k0decreases from 75 s−1for maltose to 3 s−1for maltitol while theKmvalue increases from 4.2 to 50 mM. Leucrose with α-1,5-glycosidic bond was hydrolyzed with ak0value of 8 s−1and aKmvalue of 74 mM. Disaccharides with α-1,6-glycosidic bonds were hydrolyzed with extremely low rates: for isomaltose and isomaltulosek0values of 5 and 3 s−1, respectively, andKmvalues of 90 and 42 mM, respectively, were observed. Again reduction lowers thek0values: The corresponding disaccharide alcohols α-d-glucopyranosyl-1,6-sorbitol and α-d-glucopyranosyl-1,6-mannitol, like isomaltooligosaccharides, were not hydrolyzed. Regarding the conformation of sucrose, leucrose, and maltose previously determined by molecular dynamics simulations, a reasonable explanation for the different rates of hydrolysis could be postulated. Based on the enzyme kinetic parameters for the series of maltooligosaccharides, subsite affinities (Ai) according to the subsite theory were calculated as 7.5 (A1), 17 (A2), 3.4 (A3), and 1.3 kJ/mol (A4) for subsites 1, 2, 3, and 4, respectively. The intrinsic rate constantkintwas estimated at 76 s−1.