A tale of two isomerases: compact versus extended active sites in ketosteroid isomerase and phosphoglucose isomerase.
A tale of two isomerases: compact versus extended active sites in ketosteroid isomerase and phosphoglucose isomerase.
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DOI：
10.1021/bi201089v
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发表时间：
2011-10
期刊：
影响因子：
2.9
通讯作者：
Srinivas Somarowthu;H. Brodkin;J. A. D'aquino;D. Ringe;M. Ondrechen;P. Beuning
中科院分区：
文献类型：
--
作者：
Srinivas Somarowthu;H. Brodkin;J. A. D'aquino;D. Ringe;M. Ondrechen;P. Beuning
了解酶的催化效率和特异性是生物化学中具有重大实践和概念重要性的基本问题。虽然生化和结构研究的进展丰富了我们对酶的了解，但在酶催化中，与反应底物分子不是最近邻的残基的作用在很大程度上是未经实验探索的。在这里，计算活性位点的预测，THEMATICS和池，用于确定功能上重要的残基，不直接接触的反应底物分子。这些预测，然后引导实验，探索两个异构酶，恶臭假单胞菌甾酮异构酶（KSI）和人类磷酸葡萄糖异构酶（PGI）的活性位点，作为原型的非常不同类型的预测活性位点。KSI和PGI都是EC 5.3的成员，催化类似的反应，但它们代表了显着不同程度的远程残基参与，如THEMATICS和POOL所预测的。对于KSI，预测主要是第一壳残基的紧凑活性位点，但是对于PGI，预测在反应底物周围的第一、第二和第三层中的残基的扩展活性位点。通过定点诱变和动力学分析研究了先前未通过实验测试的预测残基。在人PGI中，预测的第二和第三壳残基K362、H100、E495、D511、H396和Q388的单点突变显示相对于野生型的催化活性显著降低。这些实验的结果表明，正如预测的那样，远程残基在PGI催化中非常重要，但在KSI中对催化的贡献很小。
Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.