血红素加氧酶-1中第39位赖氨酸对电子传递功能的影响

The effect on the function of electron transport of the lysine 39 in heme oxygenase-1

目的验证血红素加氧酶－１（ＨＯ－１）中的第３９位赖氨酸（Ｋ３９）对电子传递的影响。方法通过定点诱变将Ｋ３９置换为酸性氨基酸谷氨酸（Ｅ），并将得到的变异型酶基因克隆到高度表达的载体上表达，提纯变异型ＨＯ，测定其活性，比较变异型酶与野生型酶的活性区别，以了解高保守的碱性氨基酸Ｋ３９对电子传递的影响。结果 ＨＯ催化血红素的体外反应，由抗坏血酸提供电子时，变异型酶Ｋ３９Ｅ与野生型酶比较活性没有显著性差异；而ＨＯ催化血红素的模拟体内反应，由ＮＡＤＰＨ－细胞色素Ｐ４５０还原酶提供电子时，变异型酶Ｋ３９Ｅ与野生型酶相比活性有显著性差异。结论 ＨＯ的碱性氨基酸Ｋ３９在接受并传递ＮＡＤＰＨ－细胞色素Ｐ４５０所提供的电子时具有重要作用。

Objective To clarify the important role of highly conserved alkaline acid the lysine 39 of heme oxygenase-1 (HO-1) in the process of electron transport. Methods The lysine 39(K39) were converted to glutamic acid (E) 39 through site-directed mutagenesis, then the mutant gene were cloned into expression vector, the mutant HO were purified and its enzyme activity were determined. Results When ascorbic acid was used as an electron donor, the enzyme activity of K39E had no significant difference from its wild type; But when NADPH-cytochrome P450 reductase was used as an electron donor, the enzyme activity of K39E had significant difference from its wild type. Conclusion The K39 in HO has important effects on accepting and transporting electrons from NADPH-cytochrome P450 reductase.