Kinetic studies on the substrate specificity and active site of rabbit muscle acid alpha-glucosidase.
Kinetic studies on the substrate specificity and active site of rabbit muscle acid alpha-glucosidase.
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兔肌酸α-葡萄糖苷酶底物特异性和活性位点的动力学研究。
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发表时间：
1984
期刊：
影响因子：
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通讯作者：
S. Chiba
中科院分区：
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作者：
H. Matsui;M. Sasaki;E. Takemasa;T. Kaneta;S. Chiba
本文首次从兔肌肉中分离纯化了哺乳动物肌肉酸性α-葡萄糖苷酶，经硫酸铵分级分离、SephadexG-100、CM-TOYOPEARL和TOYOPEARLHW-55柱层析，得到了高纯度的酶。所得制剂在聚丙烯酰胺圆盘凝胶电泳上显示单一条带。SDS-电泳测得其分子量为1.02 × 10 ~（5）。最适pH为4.5。α-葡糖苷酶不仅对麦芽糖，而且对α-葡聚糖，如可溶性淀粉、β-极限糊精、支链淀粉、贝类糖原和直链淀粉显示出相对高的活性。麦芽糖和糖原的Km值分别为6.3 mM和12 mM（非还原性葡萄糖单位的浓度），两种底物水解的最大速度之比依次为100：66.7。兔肌肉酸性α-葡萄糖苷酶对各种底物具有广泛的特异性。平均聚合度为13和17的麦芽糖、麦芽三糖、β-四糖、β-五糖、β-六糖、β-七糖和β-八糖以及麦芽糊精的Km值分别为6.3 mM、2.6 mM、5.9 mM、3.0 mM、5.9 mM、5.9 mM、5.9 mM、7.7 mM和5.6 mM。由三个或三个以上葡萄糖单元组成的麦芽寡糖的相对最大速度是麦芽糖的43.5-89.3%。对于二糖，水解速率按以下顺序降低：麦芽糖除以黑曲霉糖大于曲二糖大于异麦芽糖。纯化的酶是哺乳动物来源的典型酸性α-葡萄糖苷酶，其水解各种底物以产生α-葡萄糖。用动力学方法研究了催化麦芽糖和糖原水解的活性中心的性质。在实验中与混合底物，麦芽糖和贝类糖原，动力学特征同意非常密切的理论预测为一个单一的网站机制。必需的可电离基团，1（在酸性侧）和2（在碱性侧），被确定为-COO-和-COOH的两种底物的水解。阳离子，Na+，K+，和Mg 2+，大约同样有效的酶的麦芽糖和贝类糖原的刺激作用。三羟甲基氨基甲烷、松二糖和蔗糖不仅抑制麦芽糖酶的活性，而且抑制糖化酶的活性。从这些结果可以得出结论，兔肌肉酸性α-葡糖苷酶通过单一活性位点机制攻击麦芽糖和糖原。
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.