Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine
Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine
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DOI：
10.1021/bi0262348
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发表时间：
2002-11-19
期刊：
影响因子：
2.9
通讯作者：
Dumas, R
中科院分区：
文献类型：
--
作者：
Halgand, F;Wessel, PM;Dumas, R
拟南芥苏氨酸脱氨酶（TD）是由59600个相同的Da亚基组成的四聚体。异亮氨酸抑制了TD的活性。这种效应被大量过量的缬氨酸所逆转。非变性凝胶过滤、聚丙烯酰胺凝胶电泳和质谱实验表明，异亮氨酸在TD上的结合诱导了酶的二聚化，而高缬氨酸浓度的加入则恢复了酶的四聚化。在异亮氨酸增加的情况下，非变性凝胶过滤和电喷雾电离质谱分析表明，四聚体和二聚体之间存在快速平衡。最后，对TD突变体的研究使我们能够关注每个异亮氨酸结合位点的具体作用。
Arabidopsis thaliana threonine deaminase (TD) is a tetramer composed of identical similar to59600 Da subunits. TD activity has been shown to be inhibited by isoleucine. This effect is reversed by a large excess of valine. Nondenaturant gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry experiments demonstrated that binding of isoleucine on TD induces dimerization of the enzyme, whereas tetramerization is restored by addition of a high valine concentration. Nondenaturant gel filtration and electrospray ionization mass spectrometry of the enzyme in the presence of increasing amounts of isoleucine suggest a fast equilibrium between the tetramer and the dimer. Finally, study of TD mutants allowed us to focus on the specific role of each isoleucine-binding site.