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 alpha-1, beta-2-glycosidic bond of the disaccharide sucrose was not hydrolyzed. Various substrates with alpha-1,4-glycosidic bonds (maltose, maltooligosaccharides) were hydrolyzed with high maximal reaction velocities. Reduction lowered the rate of hydrolysis drastically: k'0 decreases from 75 s-1 for maltose to 3 s-1 for maltitol while the K(m) value increases from 4.2 to 50 mM. Leucrose with alpha-1,5-glycosidic bond was hydrolyzed with a k'0 value of 8 s-1 and a K(m) value of 74 mM. Disaccharides with alpha-1,6-glycosidic bonds were hydrolyzed with extremely low rates: for isomaltose and isomaltulose k'0 values of 5 and 3 s-1, respectively, and K(m) values of 90 and 42 mM, respectively, were observed. Again reduction lowers the k'0 values: The corresponding disaccharide alcohols alpha-D-glucopyranosyl-1,6-sorbitol and alpha-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 (A1) 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 constant k'int was estimated at 76 s-1.
Günther
S
S
Zentrum Biochemie, Medizinische Hochschule Hannover, Germany. sguent@botanik.tiho-hannover.de
Heymann
H
H
D000818:Animals; D001665:Binding Sites; D004187:Disaccharides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D007422:Intestines / Q000201:enzymology; D007534:Isomaltose / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D000520:alpha-Glucosidases / Q000378:metabolism*
D004187:Disaccharides; D006027:Glycosides; D009844:Oligosaccharides; D007534:Isomaltose; D008320:Maltose; D000520:alpha-Glucosidases