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首页> 外文期刊>Biochemistry >Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments: differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site.
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Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments: differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site.

机译：不同热环境中梭子鱼（Sphyraena属）的乳酸脱氢酶-A同源物的进化：动力学特性和热稳定性的差异是由于活性位点以外的氨基酸取代引起的。

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摘要

Orthologous homologs of lactate dehydrogenase-A (LDH-A) (EC 1.1.1.27; NAD+:lactate oxidoreductase) of six barracuda species (genus Sphyraena) display differences in Michaelis-Menten constants (apparent Km) for substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in Km with increasing measurement temperature occur for all homologs, yet Km at normal body temperatures is similar among species because of the inverse relationship between adaptation temperature and Km. Thermal stabilities of the homologs also differ. To determine the amino acid substitutions responsible for differences in Km and thermal stability, peptide mapping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar peptide maps, those of the north temperate species, S. argentea, the subtropical species, S. lucasana, and the south temperate species, S. idiastes, were deduced from the respective cDNAsequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzymes. The sequence of LDH-A from S. lucasana differs from that of S. idiastes only at position 8. The homolog of S. argentea differs from the other two sequences at positions 8, 61, 68, and 223. We used a full-length cDNA clone of LDH-A of S. lucasana to test, by site-directed mutagenesis, the importance of these sequence changes in establishing the observed differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences in Km between the LDH-As of S. argentea and S. lucasana. Differences at position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary adaptation of proteins to temperature thus may be achieved by minor changes in sequence at locations outside of active sites, and these changes may independently affect kinetic properties and thermal stabilities.
机译：六个梭子鱼（Sphyraena属）的乳酸脱氢酶-A（LDH-A）（EC 1.1.1.27; NAD +：乳酸氧化还原酶）的直系同源物显示底物（丙酮酸）和辅因子的Michaelis-Menten常数（表观Km）有所不同NADH）反映了不同栖息地温度下的进化。所有同系物均会随着测量温度的升高而使Km显着增加，但由于适应温度与Km之间呈反比关系，因此物种间正常体温下的Km相似。同源物的热稳定性也不同。为了确定负责Km和热稳定性差异的氨基酸取代，首先对所有六个物种的LDH-As进行了肽图分析。然后，从图谱中推导了三个具有最相似肽图的同系物的氨基酸序列，分别是北温带种，链球菌，亚热带种，卢卡萨纳和南温带种的菌丝。各自的cDNA序列。在任何一对物种之间最多有四个氨基酸取代，这些都没有发生在酶的环或底物结合位点。来自卢卡萨链霉菌的LDH-A序列仅与第8位的idiastes链霉菌序列不同。Argentea链霉菌的同系物与第8、61、68和223位的其他两个序列不同。链球菌LDH-A的全长cDNA克隆通过定点诱变来测试这些序列改变在建立观察到的动力学和热稳定性差异中的重要性。在位点61和/或68处的序列差异似乎是造成S. argentea和S. lucasana的LDH-As之间的Km差异的原因。位置8上的差异似乎解释了阿根廷链球菌和卢卡萨链霉菌的同系物之间热稳定性的差异。因此，蛋白质对温度的进化适应性可以通过在活性位点之外的位置处的序列的微小变化来实现，并且这些变化可以独立地影响动力学特性和热稳定性。

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