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首页> 外文期刊>Biochemistry >Structural Basis for a Change in Substrate Specificity: Crystal Structure of S113E Isocitrate Dehydrogenase in a Complex with Isopropylmalate, Mg~(2+), and NADP
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Structural Basis for a Change in Substrate Specificity: Crystal Structure of S113E Isocitrate Dehydrogenase in a Complex with Isopropylmalate, Mg~(2+), and NADP

机译：底物特异性变化的结构基础：S113E乙酸盐脱氢酶的晶体结构在络合物中的异丙基酸盐，Mg〜（2+）和NADP

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摘要

Isocitrate dehydrogenase (JDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The 811 3E mutant of JDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (kcat/KM) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH 811 3E in a complex with isopropylmalate, NADP, and Mg2± to 2.0 A resolution. On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates. The 811 3E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site. In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the 811 3E substitution and the binding of substrates act together to promote catalysis of isopropylmalate. Ligand binding results in reorientation of the active site helix that contains residues 113 through 116. El 13 exhibits new interactions, including van der Wuals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with NI 15, which in turn forms a hydrogen bond with NADP. In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme. In combination, these interactions appear to provide the basis for the switch in substrate specificity.
机译：异柠檬酸脱氢酶（JDH）催化苯乙酸的氧化脱羧，偏离其他（R）的型衬底具有可忽略的活性。 JDH的811 3E突变体显着提高了其利用异丙基丙酸酯作为基材的能力，并将衬底特异性（KCAT / Km）从异柠檬酸酯切换为异丙基丙酸盐。为了了解底物特异性的该开关的结构基础，我们已经确定了IDH 811 3E的晶体结构，其在络合物中与异丙基丙酸酯，NADP和MG2±2.0分辨率。在与先前确定的结构的比较的基础上，我们确定由氨基酸取代和底物结合引起的明显变化。与其他IDH结构相比，811 3E复合物在全局和有源站点构象中展示了改变，其包括活动位点附近的循环和螺旋构象变化的其他IDH结构。另外，铰链的角度在该结构中改变了与两个结构域改变，这表明811 3E取代和底物的结合作用在一起以促进异丙基丙酸盐的催化。配体结合导致含有残留物113至116的活性位点螺旋的重新定位。EL 13表现出新的相互作用，包括van der Wuals与异丙基丙酸盐的异丙基和Ni 15的氢键，其又形成氢键NADP。另外，改变结合NADP的环和螺旋区域，与将NADP结合区域连接到有源位点螺旋的环，改变底物和酶之间的关系。组合，这些相互作用似乎为开关底物特异性提供了基础。

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  • 来源
    《Biochemistry》 |2001年第14期|共8页
  • 作者

    Sharon A. Doyle; Peter T. Beernink; Daniel E. Koshland;

  • 作者单位

    Department of Molecular and Cell Biology University of California at Berkeley Berkeley California 94720 and Molecular and Structural Biology Division Lawrence Livermore National Laboratory 7000 East Avenue L-448 Livermore California 94550;

    Department of Molecular and Cell Biology University of California at Berkeley Berkeley California 94720 and Molecular and Structural Biology Division Lawrence Livermore National Laboratory 7000 East Avenue L-448 Livermore California 94550;

    Department of Molecular and Cell Biology University of California at Berkeley Berkeley California 94720 and Molecular and Structural Biology Division Lawrence Livermore National Laboratory 7000 East Avenue L-448 Livermore California 94550;

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  • 正文语种 eng
  • 中图分类 生物化学;
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