作　　者： ; ; (洪钧言）;

机构地区： 深圳大学生命科学学院

出　　处： 《中山大学学报（医学科学版）》 2009年第2期126-131,共6页

摘　　要： 【目的】通过比较人体主要尼古丁代谢酶CYP2A6和CYP2A13野生型及突变体酶蛋白对香豆素7-羥化反应的催化动力学特征,确定影响两者催化活性差异的关键氨基酸残基,提供建立评估人群尼古丁代谢酶基因多态性与吸烟行为及癌症易感性生物标记的依据。【方法】运用定点突变和Sf9昆虫杆状病毒蛋白表达体系产生CYP2A6和CYP2A13在300,301以及369位点氨基酸互换的突变体蛋白质,测定系列香豆素浓度下的各个酶蛋白对香豆素7-羥化的催化反应活性,获得动力学参数并与野生型酶蛋白进行比较分析。【结果】与野生型CYP2A6相比较,其Ile300→Phe突变体催化效率(Kcat=Vmax/Km)显著下降而Gly301→Ala突变体催化效率显著上升(1.64和8.02相对于野生型3.56,P<0.01)。与之相对应,CYP2A13的Phe300→Ile突变体催化效率则明显上升而Ala301→Gly突变体催化效率显著下降(1.03和0.06相对于野生型的0.27,P<0.05)。这些酶催化特性的改变,主要是由于Vmax的改变所致。369位的氨基酸残基,无论是Ser还是Gly对CYP2A6或CYP2A13的香豆素7-羥化反应催化活性均无明显的影响。【结论】300和301位的氨基酸是影响CYP2A6及CYP2A13对香豆素7-羥化反应催化活性的关键残基,而369位氨基酸不影响此活性。 [Objective] To compare the catalytic kinetic characteristics of major human nicotine metabolic enzymes--the wildtype CYP2A6 and CYP2A13 and the enzyme protein of their mutants in coumarin 7-hydrocylation, to identify the key amino acid residues that influence their catalytic activities, and to support fundamental data to develop biomarkers for the assessment of CYP2A gene polymorphism, smoking behavior, and cancer susceptibility. [Methods] A set of mutants with reciprocal substitution of amino acid residues at 300, 301 and 369 positions of CYP2A6 and CYP2A13 were generated by point mutation and the insect baculovirus protein expression system. The catalytic reaction activities of all enzyme proteins were determined at different coumarin concentrations and kinetic parameters were obtained to compare with that of wild-type enzyme proteins. [Results] Compared with that of wild-type CYP2A6, the catalytic efficiency （Kcat =Vmax/Km） of Ile^300→Phe mutant decreased significantly, while that of Gly^301→ Ala mutant increased significantly （Kcat of 1.64 and 8.02 vs. 3.56 of the wild-type CYP2A6, P 〈 0.01）. Correspondently, the catalytic efficiency of CYP2A13 Phe^300→ Ile mutant increased, while that of CYP2A13 Ala^301→Gly mutant decreased remarkably （Kcat of 1.03 and 0.06 vs.0.27 of the wild-type CYP2A13, P 〈 0.05）. The changes of the catalytic characteristics were due to the changes of Vmax. Amino acid residues Ser or Gly at position 369 in either CYP2A6 or CYP2A13 did not show significant influence on the enzyme catalytic efficiency in coumarin 7-hydroxlation. [Conclusions] Amino acid residues at position 300 and 301 are the key residues in both CYP2A6 and CYP2A13 that affect the catalytic activities of eoumarin 7-hydroxylation. However, the amino acid residues at position 369 do not affect the catalytic activities of coumarin metabolism.

关 键 词： 点突变 香豆素代谢 关键氨基酸

领　　域： [生物学]