HUMAN MICROSOMAL EPOXIDE HYDROLASE - GENETIC-POLYMORPHISM AND FUNCTIONAL EXPRESSION IN-VITRO OF AMINO-ACID VARIANTS
HUMAN MICROSOMAL EPOXIDE HYDROLASE - GENETIC-POLYMORPHISM AND FUNCTIONAL EXPRESSION IN-VITRO OF AMINO-ACID VARIANTS
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DOI：
10.1093/hmg/3.3.421
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发表时间：
1994-03-01
影响因子：
3.5
通讯作者：
OMIECINSKI, CJ
中科院分区：
文献类型：
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作者：
HASSETT, C;AICHER, L;OMIECINSKI, CJ
人微粒体环氧化物水解酶（mEH）是一种生物转化酶，可将活性环氧化物中间体代谢为水溶性更高的反式二氢二醇衍生物。我们比较了来自6个全长人mEH DNA克隆的蛋白质编码序列，并评估了7个位置的潜在氨基酸变异。使用聚合酶链反应实验在至少37个无关个体中评估这些变体的患病率。仅观察到Tyr/His 113（外显子3）和His/Arg 139（外显子4）变异体。残基113等位基因的基因型频率表明该位点可能不处于哈代-温伯格平衡，而残基139等位基因的频率与预期值相似。使用定点诱变在mEH cDNA中构建编码变体氨基酸的核苷酸序列，并通过瞬时转染COS-1细胞在体外表达每个序列。评价每个构建体的环氧化物水解酶mRNA水平、催化活性和免疫反应性蛋白。这些分析的结果证明了构建体之间相对均匀的mEH RNA表达水平。mEH酶活性和免疫反应蛋白强烈相关，表明mEH特异性活性对于每个变体是相似的。然而，显着的差异，注意到免疫反应性蛋白质和酶活性的相对量所产生的氨基酸取代。这些数据表明，常见的人类mEH氨基酸多态性可能会改变酶的功能，可能通过修改蛋白质的稳定性。
Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy - Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability.