Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase.
Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase.
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发表时间：
1989-01
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影响因子：
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通讯作者：
M. Tagaya;T. Yagami;T. Noumi;M. Futai;F. Kishi;A. Nakazawa;T. Fukui
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文献类型：
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作者：
M. Tagaya;T. Yagami;T. Noumi;M. Futai;F. Kishi;A. Nakazawa;T. Fukui
通过定点诱变，腺苷酸激酶富含甘氨酸的区域中的脯氨酸 17 被 Gly（Gly 突变体）或 Val（Val 突变体）取代。溶解大肠杆菌细胞裂解物沉淀中的蛋白质后，通过十二烷基硫酸钠凝胶电泳将突变酶纯化至均质状态。与野生型酶相比，AMP 的 Gly 突变体和 Val 突变体的表观 Km 值分别增加了约 7 倍和 24 倍。 Gly 和 Val 突变体中 ATP 的表观 Km 值也分别增加了 7 倍和 42 倍。相反，两种突变酶的 Vmax 值与野生型酶相当。这些结果表明，Pro-17 对于底物的结合起着重要作用，但对于催化效率却没有起到重要作用，尽管它不直接与底物相互作用。腺苷二磷酸吡哆醛特异性修饰腺苷酸激酶中的 Lys-21（Tagaya, M.、Yagami, T. 和 Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261），以几乎相同的速率灭活野生型和突变型酶。有趣的是，两种突变酶都比野生型酶对腺嘌呤核苷酸表现出更高的特异性。与野生型酶相比，两种突变酶对高温或胰蛋白酶处理失活的抵抗力较低。看来突变酶的大部分特性可以基于环的构象灵活性的需要来解释，其中包括用于底物结合的Pro-17。
Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding.