MONOVALENT CATIONS PARTIALLY REPAIR A CONFORMATIONAL DEFECT IN A MUTANT TRYPTOPHAN SYNTHASE ALPHA(2)BETA(2) COMPLEX (BETA-E109A)
MONOVALENT CATIONS PARTIALLY REPAIR A CONFORMATIONAL DEFECT IN A MUTANT TRYPTOPHAN SYNTHASE ALPHA(2)BETA(2) COMPLEX (BETA-E109A)
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DOI：
10.1074/jbc.270.29.17333
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发表时间：
1995-07-21
影响因子：
4.8
通讯作者：
MILES, EW
中科院分区：
文献类型：
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作者：
RUVINOV, SB;AHMED, SA;MILES, EW
我们使用色氨酸合成酶α (2) β(2)复合物作为模型系统来研究配体、蛋白质-蛋白质相互作用和突变如何调节酶活性、反应特异性和底物特异性。l -丝氨酸和吲哚单独由β(2)亚基转化为l -色氨酸的速率很低，但被某些单价阳离子激活或与α亚基结合形成α (2) β(2)复合物。由于一价阳离子和亚基似乎稳定了β(2)亚基的活性构象，我们研究了一价阳离子对α (2) β(2)复合物突变形式的活性和光谱特性的影响，β(2)亚基谷氨酸109被丙氨酸（E109A）取代。E109A α (2) β(2)复合物在与l -丝氨酸的反应中无活性，但在与-氯- l -丙氨酸的反应中有活性。平行实验显示了一价阳离子对野生型β(2)亚基和α (2) β(2)配合物性质的影响。我们发现，CsCl刺激了E109A α (2) β(2)复合物和野生型β(2)亚基与l -丝氨酸和吲哚的活性，改变了l -丝氨酸反应中间体的平衡分布。结果表明，CsCl通过稳定与野生型α (2) β(2)复合物更相似的催化性质的构象，部分修复了E109A突变对α (2) β(2)复合物活性的有害影响。这一结论与一价阳离子通过稳定替代构象改变几种吡哆醛磷酸依赖酶的催化和光谱特性的观察结果是一致的。
We are using the tryptophan synthase alpha(2) beta(2) complex as a model system to investigate how ligands, protein-protein interaction, and mutations regulate enzyme activity, reaction specificity, and substrate specificity. The rate of conversion of L-serine and indole to L-tryptophan by the beta(2) subunit alone is quite low, but is activated by certain monovalent cations or by association with alpha subunit to form an alpha(2) beta(2) complex. Since monovalent cations and a subunit appear to stabilize an active conformation of the beta(2) subunit, we have investigated the effects of monovalent cations on the activities and spectroscopic properties of a mutant form of alpha(2) beta(2) complex having beta(2) subunit glutamic acid 109 replaced by alanine (E109A). The E109A alpha(2) beta(2) complex is inactive in reactions with L-serine but active in reactions with beta-chloro-L-alanine. Parallel experiments show effects of monovalent cations on the properties of wild type beta(2) subunit and alpha(2) beta(2) complex. We find that CsCl stimulates the activity of the E109A alpha(2) beta(2) complex and of wild type beta(2) subunit with L-serine and indole and alters the equilibrium distribution of L-serine reaction intermediates. The results indicate that CsCl partially repairs the deleterious effects of the E109A mutation on the activity of the alpha(2) beta(2) complex by stabilizing a conformation with catalytic properties more similar to those of the wild type alpha(2) beta(2) complex. This conclusion is consistent with observations that monovalent cations alter the catalytic and spectroscopic properties of several pyridoxal phosphate dependent enzymes by stabilizing alternative conformations.