STRUCTURE AND FUNCTION ANALYSIS OF P-450

细胞色素P450负责多种外源基因的代谢 以药物和药物的形式摄入或吸收的化合物 环境污染物。一般认为，50到200个P450 形式可以在特定的有机体中表达。每种P450形式都可以代谢 许多结构上无关的化合物。一种化合物也可以 有时被多种形式的P450代谢。经常，肯定的 化学物质被转化为高能的亲电体，它们从 P450活性部位，并可损伤DNA。我们利用了两个结果 以确定其结构与功能的关系 I)共享的密切相关的子家族成员的活跃站点 相当大的序列相似性但具有不同的催化活性， 和ii)具有不同催化活性的等位基因变体，但只有少数 氨基酸的差异。我们利用c DNA构建了嵌合的P450 操纵和表达以确定P450的哪些区域 蛋白质决定底物的特异性。使用Site进行进一步研究 将进行定向突变以确定哪种氨基酸 残基负责特定的催化活性。 我们亦正进行原址重建研究，以确定 P450形式对NADPH-P450氧化还原酶和细胞色素b5的要求 使用痘苗病毒表达系统。肝癌细胞同时感染 用含有P450、NADPH-P450氧化还原酶和 细胞色素b5cDNA。然后用完整的方法测量催化活性 细胞和亚细胞比例。

Cytochrome P450s are responsible for metabolism of numerous foreign compounds that are ingested or absorbed in the form of drugs and environmental contaminants. It is generally believed that 50 to 200 P450 forms may be expressed in a given organism. Each P450 form can metabolize numerous structurally unrelated compounds. A single compound can also sometimes be metabolized by numerous P450 forms. Frequently, certain chemicals are converted to high energy electrophiles that escape from the P450 active site and can damage DNA. We have taken advantage of two results of P450 evolution to determine the structure-function relationship of their active sites in i) closely related subfamily members that share considerable sequence similarities but have differing catalytic activities, and ii) allelic variants with differing catalytic activities but only a few amino acid differences. We have made chimeric P450s through use of cDNA manipulation and cDNA expression to determine which regions of the P450 proteins determine substrate specificities. Further studies using site directed mutagenesis will be undertaken to determine which amino acid residues are responsible for specific catalytic activities. We are also carrying out in situ reconstitution studies to determine the requirements of P450 forms for NADPH-P450 oxidoreductase and cytochrome b5 using the vaccinia virus expression system. Hepatoma cells are coinfected with recombinant vaccinia containing P450, NADPH-P450 oxidoreductase and cytochrome b5 cDNAs. Catalytic activities are then measured using intact cells and subcellular fraction.