Kinetic studies on the substrate specificity and active site of rabbit muscle acid alpha-glucosidase.
Mammalian muscle acid alpha-glucosidase was highly purified for the first time from rabbit muscle by fractionation with ammonium sulfate, and chromatographies on Sephadex G-100, CM-TOYOPEARL and TOYOPEARL HW-55. The resulting preparation showed a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be 1.02 X 10(5) by SDS-electrophoresis. The optimum pH was found to be 4.5. The alpha-glucosidase showed relatively high activity not only toward maltose but also toward alpha-glucans, such as soluble starch, beta-limit dextrin, amylopectin, shellfish glycogen, and amylose. The Km values for maltose and glycogen were 6.3 mM and 12 mM (the concentration of non-reducing glucose units), respectively, and the ratio of the maximum velocities of hydrolyses of the two substrates was 100:66.7, in that order. Rabbit muscle acid alpha-glucosidase showed a wide specificity for various substrates. The Km values for maltose, maltotriose, -tetraose, -pentaose, -hexaose, -heptaose, and -octaose, and maltodextrins of average polymerization degrees of 13 and 17 were 6.3 mM, 2.6 mM, 5.9 mM, 3.0 mM, 5.9 mM, 5.9 mM, 5.9 mM, 7.7 mM, and 5.6 mM, respectively. The relative maximum velocities for maltooligosaccharides consisting of three or more glucose units were 43.5-89.3% of that for maltose. For disaccharides, the rate of hydrolysis decreased in the following order: maltose divided by nigerose greater than kojibiose greater than isomaltose. The purified enzyme was a typical acid alpha-glucosidase of mammalian origin, which hydrolyzed various substrates to produce alpha-glucose. The nature of the active site catalyzing the hydrolyses of maltose and glycogen was investigated by some kinetic methods. In experiments with mixed substrates, maltose and shellfish glycogen, the kinetic features agreed very closely with those theoretically predicted for a single site mechanism. The essential ionizable groups, 1 (on the acidic side) and 2 (on the alkaline side), were identified as -COO- and -COOH for the hydrolysis of both substrates. Cations, Na+, K+, and Mg2+, were about equally effective for stimulation of the enzyme actions on maltose and shellfish glycogen. Tris, turanose and erythritol inhibited not only maltase activity but also glucoamylase activity of the enzyme. From these results, it was concluded that rabbit muscle acid alpha-glucosidase attacks maltose and glycogen by a single active site mechanism.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D005959:Glucosidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D013045:Species Specificity; D013379:Substrate Specificity; D013816:Thermodynamics; D000520:alpha-Glucosidases / Q000302:isolation & purification / Q000378:metabolism*
D005959:Glucosidases; D000520:alpha-Glucosidases