The mechanism of action of sucrose phosphorylase. Isolation and properties of a beta-linked covalent glucose-enzyme complex.
The mechanism of action of sucrose phosphorylase. Isolation and properties of a beta-linked covalent glucose-enzyme complex.
复制标题
蔗糖磷酸化酶的作用机制。
DOI：
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发表时间：
1970
影响因子：
4.8
通讯作者：
R. Abeles
中科院分区：
文献类型：
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作者：
J. Voet;R. Abeles
当蔗糖磷酸化酶与蔗糖反应时，可分离出共价葡萄糖-酶复合物。在一种情况下，通过在酸性条件下快速使酶变性来分离络合物。在另一种情况下，通过添加底物后用NaIO4对蛋白质进行化学修饰，分离出配合物。naio4修饰的复合物保持了将葡萄糖基转移到受体的能力，具有与天然酶相似的受体特异性。反应速率约为天然酶的1/20,000。胃蛋白酶消化这两种复合物会产生带正电荷的含葡萄糖片段，可能是多肽。葡萄糖和多肽之间以及葡萄糖和酶之间的连接是非常不稳定的。对于葡萄糖基肽，在pH 6.0时，在25°温度下，葡萄糖的释放时间为80分钟。当葡萄糖在75%的甲醇中从葡萄糖基肽中释放出来时，只生成葡萄糖而不生成甲基葡萄糖苷。结论是葡萄糖必须通过肽提供的氧原子与肽连接，并且C-1葡萄糖氧键在溶解过程中没有断裂。从肽中释放的葡萄糖具有β构型。因此，葡萄糖-酶复合物的形成是由蔗糖的糖基部分C-1原子的构型反转进行的。这一事实，再加上葡萄糖-酶复合物的分离，为蔗糖磷酸化酶通过双重替代机制发挥作用的假设提供了支持，其中形成了β-连接的葡萄糖-酶复合物。
Abstract When sucrose phosphorylase reacts with sucrose, a covalent glucose-enzyme complex can be isolated. In one case, the complex was isolated by rapidly denaturing the enzyme under acidic conditions. In the other case, the complex was isolated by chemically modifying the protein with NaIO4 after addition of the substrate. The NaIO4-modified complex maintains the ability to transfer the glucosyl group to acceptors with an acceptor specificity that resembles the native enzyme. The rate of reaction is approximately 1/20,000 that of the native enzyme. Pepsin digestion of both types of complexes produces positively charged, glucose-containing fragments, presumably peptides. The linkage between glucose and peptides as well as between glucose and enzyme is extremely base labile. For the glucosyl-peptides, t½ of glucose release at pH 6.0 is 80 min at 25°. When glucose is released from glucosyl peptides in 75% methanol, only glucose and no methyl glucoside is formed. It was concluded that glucose must be linked to the peptide through an oxygen atom contributed by the peptide and that the C-1 glucose oxygen bond was not broken during solvolysis. The glucose released from the peptides has the β configuration. Therefore, formation of the glucose-enzyme complex proceeds with inversion of configuration at the C-1 atom of the glucosyl moiety of sucrose. This fact, together with the isolation of a glucose-enzyme complex, provides support for the hypothesis that sucrose phosphorylase functions through a double replacement mechanism with intermediate formation of a β-linked glucose-enzyme complex.