Structural basis for a change in substrate specificity:: Crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP
Structural basis for a change in substrate specificity:: Crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP
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DOI：
10.1021/bi002533q
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发表时间：
2001-04-10
期刊：
影响因子：
2.9
通讯作者：
Koshland, DE
中科院分区：
文献类型：
--
作者：
Doyle, SA;Beernink, PT;Koshland, DE
异柠檬酸脱氢酶（IDH）催化异柠檬酸的氧化脱羧作用，对其他（R）-苹果酸型底物的活性可忽略不计。IDH的S113 E突变体显著提高了其利用苹果酸异丙酯作为底物的能力，并将底物特异性（k（cat）/K-M）从异柠檬酸转换为苹果酸异丙酯。为了理解这种底物特异性转换的结构基础，我们确定了晶体。IDH S113 E与苹果酸异丙酯、NADP和Mg 2+的复合物的结构，分辨率为2.0埃。在与先前确定的结构进行比较的基础上，我们确定了由氨基酸取代和底物结合引起的不同变化。与其他IDH结构相比，S113 E复合物在全球和活性位点构象中表现出改变，所述其他IDH结构包括活性位点附近的环和螺旋构象变化。此外，在这种结构中，与两个结构域相关的铰链的角度发生了改变，这表明S113 E取代和底物的结合一起起作用以促进苹果酸异丙酯的催化。配体结合导致包含残基113至116的活性位点螺旋的重新取向。E113表现出新的相互作用，包括与苹果酸异丙酯的异丙基的货车范德华接触和与N115的氢键，N115又与NADP形成氢键。此外，结合NADP的环和螺旋区域被改变，连接NADP结合区域和活性位点螺旋的环也被改变，改变了底物和酶之间的关系。结合起来，这些相互作用似乎提供了底物特异性转换的基础。
Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates. The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K-M) from isocitrate to isopropylmalate. To understand the structural basis for this switch in substrate specificity, we have determined the crystal. structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg2+ to 2.0 Angstrom 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 S113E 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 S113E 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. E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, 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.