a Department of Biological Sciences , Konkuk University , Seoul , Korea.
J Toxicol Environ Health A. 2018;81(12):493-501. doi: 10.1080/15287394.2018.1460784. Epub 2018 Apr 13.
Humans possess three cytochrome P450 enzymes in the 2A subfamily (2A6, 2A7, and 2A13). P450 2A13 is mainly expressed in the human trachea and lung, whereas P450 2A6 is found in human liver. The P450 2A13 enzyme may be considered as the primary enzyme responsible for metabolic activation of many tobacco-specific carcinogens. Genetic variations significantly influence the toxicological consequences attributed to tobacco smoking. The aim of this study was to examine the in vitro functional activities of five P450 2A13 genetic variations (R257C, 133_134insT, R101Q, I331T, and R257C/I331T) in P450 2A13*2, *3, *4, and *10 alleles. Mutant clones were constructed and their recombinant enzymes were expressed in Escherichia coli. P450 2A13 mutants containing R257C, 133_134insT, I331T, and R257C/I331T displayed P450 holoenzyme spectra. The R101Q mutant was not apparently expressed. P450 2A13 enzymes displayed the typical type I binding spectra to coumarin and the calculated binding affinities of R257C, R257C/I331T, and 133_134insT mutants were decreased approximately three- to sevenfold. In catalytic analyses of purified mutant enzymes for coumarin and nicotine, the R257C and I331T mutants exhibited lower k values with catalytic efficiencies reduced up to approximately 20%. The double mutation of R257C/I331T induced increased K values and diminished k values that resulted in >50% decrease in catalytic efficiencies. For 133_134insT mutant, catalytic activities were not markedly saturated but the measured rates at the highest concentrations were significantly lower than those of the wild-type or other mutant enzymes. Functional analysis of these variations in P450 2A13 allelic variants may help to understand the consequences of P450 2A13 polymorphism in bioactivation of many tobacco-derived carcinogens.
人类在 2A 亚家族中拥有三种细胞色素 P450 酶（2A6、2A7 和 2A13）。P450 2A13 主要在人体气管和肺部表达，而 P450 2A6 则存在于人体肝脏中。P450 2A13 酶可能被认为是负责许多烟草特异性致癌物质代谢激活的主要酶。遗传变异会显著影响归因于吸烟的毒理学后果。本研究的目的是研究五个 P450 2A13 遗传变异（R257C、133_134insT、R101Q、I331T 和 R257C/I331T）在 P450 2A13*2、*3、4 和10 等位基因中的体外功能活性。构建了突变克隆，并在大肠杆菌中表达了它们的重组酶。含有 R257C、133_134insT、I331T 和 R257C/I331T 的 P450 2A13 突变体显示出 P450 全酶光谱。R101Q 突变体显然没有表达。P450 2A13 酶对香豆素显示出典型的 I 型结合光谱，并且 R257C、R257C/I331T 和 133_134insT 突变体的计算结合亲和力降低了约三到七倍。在对纯化突变酶的香豆素和尼古丁的催化分析中，R257C 和 I331T 突变体表现出较低的 k 值，催化效率降低了约 20%。R257C/I331T 的双重突变导致 K 值增加和 k 值降低，从而导致催化效率降低超过 50%。对于 133_134insT 突变体，催化活性没有明显饱和，但在最高浓度下测量的速率明显低于野生型或其他突变酶的速率。对 P450 2A13 等位变体中这些变异的功能分析可能有助于理解 P450 2A13 多态性在许多烟草衍生致癌物质生物活化中的后果。
