Kimura S, Smith H H, Hankinson O, Nebert D W
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD 20892.
EMBO J. 1987 Jul;6(7):1929-33. doi: 10.1002/j.1460-2075.1987.tb02453.x.
Two benzo[a]pyrene-resistant mutant clones (c1 and c37) of the mouse hepatoma Hepa-1 wild-type (wt) cell line were examined for their lack of P(1)450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)] activity. From lambda gt11 cDNA libraries, the nearly full-length P(1)450 cDNAs of wt, c1 and c37 were isolated and sequenced. The c1 cDNA was found to have a single mutation leading to premature termination of the protein after Asn-414; a rapidly migrating band corresponding to this truncated protein was found on Western immunoblots. The c37 cDNA was found to have two point mutations, leading to Leu-118----Arg-118 and Arg-245----Pro-245, but otherwise to encode the normal (524-residue) protein; the mature protein was confirmed by Western blot analysis. P(1)450 cDNA from wt, c1 and c37 and chimeric cDNAs between wt and c37 were inserted into the expression vector pAAH5 and expressed in Saccharomyces cerevisiae strain 50.L4. The Leu-118----Arg-118 mutation alone was found to have negligible effect on AHH activity, while the Arg-245----Pro-245 mutation alone leads to a 2- to 3-fold decrease in enzyme activity. The two mutations together totally abrogate AHH activity. The biologic mutant c37 provides the first evidence for the importance of Arg-245, and the complementary function of Leu-118, in normal P(1)450 enzymic function. This alteration in a single amino acid from arginine to proline might block electron flow directly, or change secondary structure of the protein, such that normal monooxygenation of benzo[a]pyrene cannot occur.
对小鼠肝癌Hepa-1野生型（wt）细胞系的两个抗苯并[a]芘突变克隆（c1和c37）进行了检测，以确定它们缺乏P(1)450[芳烃（苯并[a]芘）羟化酶（AHH）]活性。从λgt11 cDNA文库中，分离并测序了wt、c1和c37的近乎全长的P(1)450 cDNA。发现c1 cDNA有一个单突变，导致蛋白质在Asn-414后提前终止；在Western免疫印迹上发现了一条与这种截短蛋白相对应的快速迁移条带。发现c37 cDNA有两个点突变，导致Leu-118变为Arg-118以及Arg-245变为Pro-245，但除此之外编码正常的（524个残基）蛋白；成熟蛋白通过Western印迹分析得到证实。将wt、c1和c37的P(1)450 cDNA以及wt和c37之间的嵌合cDNA插入表达载体pAAH5，并在酿酒酵母菌株50.L4中表达。发现单独的Leu-118变为Arg-118突变对AHH活性影响可忽略不计，而单独的Arg-245变为Pro-245突变导致酶活性降低2至3倍。这两个突变共同完全消除了AHH活性。生物学突变体c37为Arg-245以及Leu-118在正常P(1)450酶功能中的互补作用的重要性提供了首个证据。单个氨基酸从精氨酸变为脯氨酸的这种改变可能直接阻断电子流，或改变蛋白质的二级结构，从而使苯并[a]芘无法正常进行单加氧反应。
