Effect of pH on Hydride Transfer by Escherichia coli Dihydrofolate Reductase
Effect of pH on Hydride Transfer by Escherichia coli Dihydrofolate Reductase
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DOI：
10.1002/cbic.201000794
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发表时间：
2011-05-16
期刊：
影响因子：
3.2
通讯作者：
Allemann, Rudolf K.
中科院分区：
文献类型：
--
作者：
Loveridge, E. Joel;Allemann, Rudolf K.
在由大肠杆菌二氢叶酸还原酶（EcDHFR）催化的反应中，动力学同位素效应（KIE）对氢化物转移的影响已知在pH值为7时是温度依赖性的，但在pH值升高时基本上与温度无关。在这里，我们发现从温度依赖性到温度非依赖性的转变发生在pH值为7.5和8之间。氢化物转移的活化能与pH无关。导致KIEs行为变化的机制尚不清楚，但可能涉及高pH下关键残基（或残基）去质子化引起的酶的构象变化。低pH下氢化物转移的KIE表明，在这些条件下反应的速率常数不受酶的构象变化的限制。pH对EcDHFR催化氢化物转移速率常数和KIEs的温度依赖性的影响表明，酶的运动和构象变化并不直接影响化学反应，但反应条件影响了反应前酶的构象集合，并通过这一途径控制了反应。
The kinetic isotope effect (KIE) on hydride transfer in the reaction catalysed by dihydrofolate reductase from Escherichia coli (EcDHFR) is known to be temperature dependent at pH 7, but essentially independent of temperature at elevated pH. Here, we show that the transition from the temperature-dependent regime to the temperature-independent regime occurs sharply between pH 7.5 and 8. The activation energy for hydride transfer is independent of pH. The mechanism leading to the change in behaviour of the KIEs is not clear, but probably involves a conformational change in the enzyme brought about by deprotonation of a key residue (or residues) at high pH. The KIE on hydride transfer at low pH suggests that the rate constant for the reaction is not limited by a conformational change to the enzyme under these conditions. The effect of pH on the temperature dependence of the rate constants and KIEs for hydride transfer catalysed by EcDHFR suggests that enzyme motions and conformational changes do not directly influence the chemistry, but that the reaction conditions affect the conformational ensemble of the enzyme prior to reaction and control the reaction though this route.