DISACCHARIDE AND OLIGOSACCHARIDE SUBSTRATE SPECIFICITIES AND SUBSITE BINDING-ENERGIES OF PIG INTESTINAL GLUCOAMYLASE-MALTASE

The substrate specificity of pig intestinal glucoamylase-maltase was i nvestigated. The alpha-1,beta-2-glycosidic bond of the disaccharide su crose was not hydrolyzed. Various substrates with alpha-1,4-glycosidic bonds (maltose, maltooligosaccharides) were hydrolyzed with high maxi mal reaction velocities. Reduction lowered the rate of hydrolysis dras tically: k(0)' decreases from 75 s(-1) for maltose to 3 s(-1) for malt itol while the K-m value increases from 4.2 to 50 mM. Leucrose with al pha-1,5-glycosidic bond was hydrolyzed with a k(0)' value of 8 s(-1) a nd a K-m value of 74 mM. Disaccharides with alpha-1,6-glycosidic bonds were hydrolyzed with extremely low rates: for isomaltose and isomaltu lose 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 kb values: The corresponding disaccharide alcohols alpha-D-glucopyranosy l-1,6-sorbitol and alpha-D-glucopyranosyl-1,6-mannitol, like isomaltoo ligosaccharides, were not hydrolyzed. Regarding the conformation of su crose, leucrose, and maltose previously determined by molecular dynami cs simulations, a reasonable explanation for the different rates of hy drolysis could be postulated. Based on the enzyme kinetic parameters f or the series of maltooligosaccharides, subsite affinities (Ai) accord ing to the subsite theory were calculated as 7.5 (A(1)), 17 (A(2)), 3. 4 (A(3)), and 1.3 kJ/mol (A(4)) for subsites 1, 2, 3, and 4, respectiv ely. The intrinsic rate constant k(int)' was estimated at 76 s(-1). (C ) 1998 Academic Press.