Glutamate racemase dimerization inhibits dynamic conformational flexibility and reduces catalytic rates.
Glutamate racemase dimerization inhibits dynamic conformational flexibility and reduces catalytic rates.
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DOI：
10.1021/bi9005072
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发表时间：
2009-07-28
期刊：
影响因子：
2.9
通讯作者：
Johnson, Michael E.
中科院分区：
文献类型：
--
作者：
Mehboob, Shahila;Guo, Liang;Fu, Wentao;Mittal, Anuradha;Yau, Tiffany;Truong, Kent;Johlfs, Mary;Long, Fei;Fung, Leslie W. -M.;Johnson, Michael E.
谷氨酸消旋酶（RacE）是一种将L-谷氨酸转化为D-谷氨酸的细菌酶，D-谷氨酸是肽聚糖合成的必需前体。在先前的工作中，我们已经表明两种同工型与D-谷氨酸盐共结晶为二聚体，并且酶处于封闭构象，限制了对活性位点的接近[May等人，等人（2007）J. Mol. 371，1219-1237]。RacE 2的活性位点特别受限制。我们现在利用几种计算和实验方法来了解催化过程中，当配体进入和产品退出的活性位点的整体构象动力学。我们的操纵分子动力学模拟和正常模式分析结果表明，单体形式的酶比天然二聚体形式更灵活。这些结果表明，单体酶可能比二聚体形式更有活性。因此，我们产生了破坏二聚化的位点特异性突变，并发现它们在D-Glu至L-Glu反应方向上表现出比天然酶显著更高的催化速率。从溶液X-射线散射研究恢复的低分辨率模型与从NMA获得的酶的二聚体形式的前六个正常模式良好相关。因此，沿着局部活性位点残基，全局结构域运动似乎与该酶的催化相关结构动力学有关，并表明增加的灵活性可加速催化。这是一个新的观察，远离催化位点的残基通过形成二聚体结构抑制催化活性。
Glutamate racemase (RacE) is a bacterial enzyme that converts L-glutamate to D-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms co-crystallize with D-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May et al., (2007) J. Mol. Biol. 371, 1219–1237]. The active site of RacE2 is especially restricted. We now utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and product exits the active site. Our steered molecular dynamics simulations and normal mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization, and find that they exhibit significantly higher catalytic rates in the D-Glu to L-Glu reaction direction than the native enzyme. Low resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme, and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure.
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影响因子：
2.9
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