The Temperature Dependence of the Kinetic Isotope Effects of Dihydrofolate Reductase from Thermotoga maritima Is Influenced by Intersubunit Interactions
The Temperature Dependence of the Kinetic Isotope Effects of Dihydrofolate Reductase from Thermotoga maritima Is Influenced by Intersubunit Interactions
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DOI：
10.1021/bi100761x
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发表时间：
2010-06-29
期刊：
影响因子：
2.9
通讯作者：
Allemann, Rudolf K.
中科院分区：
文献类型：
--
作者：
Loveridge, E. Joel;Allemann, Rudolf K.
来自超嗜热栖热袍菌海栖热袍菌的二氢叶酸还原酶（TmDHFR）在结构特征为染色体的DHFR中是独特的，因为它形成稳定的同源二聚体。二聚化被认为在TmDHFR的高热稳定性中起关键作用，这反映在超过85 ℃的熔融温度中。TmDHFR的二聚体界面由疏水核心和周围的带电残基组成。特别地，每个亚基的Lys 129与另一个亚基的Glu 136和Glu 138形成三元盐桥。为了探测这些盐桥在TmDHFR的二聚化和热稳定性中的作用，我们产生了一系列变体（TmDHFR-K129 E、TmDHFR-E136 K、TmDHFR-E138 K和TmDHFR-E136 K/E138 K），其中这些残基被替换为侧链带有相反电荷的残基。我们的研究结果表明，这些盐桥是TmDHFR的高热稳定性的关键，但不是二聚化的要求。虽然TmDHFR还原二氢叶酸的速率不受K129-E136-E138盐桥损失的显著影响，但观察到动力学同位素效应对氢化物转移的温度依赖性的变化。这些变化与DHFR催化可能受到酶构象整体变化的影响，而不仅仅是蛋白质运动与反应坐标的耦合的提议一致。
Dihydrofolate reductase from the hyperthermophile Thermotoga maritima (TmDHFR) is unique among structurally characterized chromosomal DHFRs in that it forms a stable homodimer. Dimerization is believed to play a key role in the high thermal stability of TmDHFR, which is reflected in a melting temperature in excess of 85 degrees C. The dimer interface of TmDHFR is composed of a hydrophobic core with charged residues around the periphery. In particular, Lys129 of each subunit forms three-membered salt bridges with Glu136 and Glu138 of the other subunit. To probe the role of these salt bridges in the dimerization and thermal stability of TmDHFR, we generated a series of variants (TmDHFR-K129E, TmDHFR-E136K, TmDHFR-E138K, and TmDHFR-E136K/E138K) in which these residues were exchanged for residues whose side chains bear the opposite charge. Our results indicate that these salt bridges are key for the high thermal stability of TmDHFR but are not a requirement for dimerization. Although the rate of dihydrofolate reduction by TmDHFR is not significantly affected by the loss of the K129-E136-E138 salt bridges, changes to the temperature dependence of the kinetic isotope effect on hydride transfer are observed. These changes are in agreement with the proposal that DHFR catalysis may be affected by changes to the conformational ensemble of the enzyme rather than only to the coupling of protein motions to the reaction coordinate.