Nature of hydrogen transfer in soybean lipoxygenase 1: Separation of primary and secondary isotope effects
Nature of hydrogen transfer in soybean lipoxygenase 1: Separation of primary and secondary isotope effects
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DOI：
10.1021/bi990834y
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发表时间：
1999-09-21
期刊：
影响因子：
2.9
通讯作者：
Klinman, JP
中科院分区：
文献类型：
--
作者：
Rickert, KW;Klinman, JP
以前对大豆脂肪氧合酶1氧化亚油酸动力学的测量表明，有很大的氚同位素效应，但不能区分初级同位素效应和cc次要效应。为了解决这个问题，我们制备了单氢代亚油酸，并用酶本身进行了对映体拆分。对初级氚同位素效应的非竞争性测量给出的值约为40，与温度无关。活化热较低且不依赖于同位素，对Arrhenius前因子有较大的同位素效应。测量到了一个非常大的明显的次生同位素效应(约2.1)，但在主要位置上测量到了一个非常小的值(1.1)，而在主要位置上使用了钚。活性中心的突变导致k(CAT)和扰动同位素效应的显著降低，特别是当氘处于主要位置时，次要效应为5.6。反常的二次同位素效应是由于氢提取的立体选择性不理想引起的，对于突变体来说，这是反向底物结合和活性碳上增加的灵活性的组合。经过修正后，对突变型和野生型酶都计算了非常大的初级(76-84)和较小的次级(1.1-1.2)动力学同位素效应。证据的份量被认为是氢隧道作为氢转移的主要机制。
Previous measurements of the kinetics of oxidation of linoleic acid by soybean lipoxygenase 1 have indicated very large deuterium isotope effects, but have not been able to distinguish the primary isotope effect from the cc-secondary effect. To address this question, singly deuterated linoleic acid was prepared, and enantiomerically resolved using the enzyme itself. Noncompetitive measurements of the primary deuterium isotope effect give a value of ca. 40 which is temperature-independent. The enthalpy of activation is low and isotope-independent, and there is a large isotope effect on the Arrhenius prefactor. A very large apparent secondary isotope effect (ca. 2.1) is measured with deuterium in the primary position, but a greatly reduced value (1.1) is observed with protium in the primary position. Mutagenesis of the active site leads to a significant reduction in k(cat) and perturbed isotope effects, in particular, a secondary effect of 5.6 when deuterium is in the primary position. The anomalous secondary isotope effects are shown to arise from imperfect stereoselectivity of hydrogen abstraction which, for the mutant, is attributed to a combination of inverse substrate binding and increased flexibility at the reactive carbon. After correction, a very large primary (76-84) and small secondary (1.1-1.2) kinetic isotope effects are calculated for both mutant and wild-type enzymes. The weight of the evidence is taken to favor hydrogen tunneling as the primary mechanism of hydrogen transfer.