REGULATION OF LIVER HEME METABOLISM & CYTOCHROME P 450 INACTIVATION

肝微粒体细胞色素P450(P450) 包括多种构成酶和诱导酶。这些单体 血红素蛋白(Mw@50 kDa)含有一种人工血红素 (铁-原卟啉IX)酶的半分子/摩尔。尽管他们 相同的血红素部分，P450在功能上不同，一种性质 由个别的血红素-细胞色素微环境赋予。P450 在各种生物的氧化/还原代谢中起着重要作用 生理上相关的内生生物和外生生物。然而， 尽管所有这些反应都很容易形成 可排泄的产品，并不都是有益的。P450催化了 某些底物对自由基和其他活性物质的代谢 可能导致毒性/病理损害的物质。此外，在 经过某些氧化还原反应，参与的P450是 在被归类为基于机制或“自杀”的过程中牺牲 失活。到目前为止，有三种不同的机制 底物介导的P450失活已被表征为：(A) 通过N-烷基/芳基化破坏假体血红素[即， 烯丙基异丙基乙酰胺(AIA)；(B)凋亡素 通过反应中间体(氯霉素，Sb， 11-十一烷酸)；和(C)破坏假体血红素以 不可逆地与细胞色素结合的产物 [CC14，螺内酯(SPL)， 3，5-dicarbethoxy-2，6-dimethyl-4-ethyl-1，4-dihydropyridine(DDEP)I.由 定义，“自杀”灭活发生在活动部位。 N-烷基化血红素HAS的分离与结构表征 明确建立了P450 N-烷基化的这一标准 亚铁血红素。然而，“自杀失活”的标准并不是 严格应用于药物诱导的P450破坏的b或c模式 因为无法进行高度的结构分析 疏水apoP450活性位点区域及其对 蛋白水解性消化(用一系列的蛋白水解酶)直到现在 这在很大程度上排除了他们明确的机械分类。vbl.使用 赖氨酰内肽酶C和/或胃酶消化以及CNBR 裂解，经血红素或药物修饰的P450肽 已经被高效液相色谱测绘和分离，作为他们 质谱学鉴定和结构表征 使用由提供的一系列质谱学技术进行分析 质谱学设备。到目前为止，两种不同的P450多肽 由血红素和Sb烷化的化合物被分离和表征，使用 ESMS和MSLDIMS。这种结构特征将极大地 有助于对模式b和模式c进行明确的机械解释 失活过程。

The hepatic microsomal hemoproteins cytochromes P450 (P450) include multiple constitutive and inducible enzymes. These monomeric hemoproteins (MW @ 50kDa) contain one prosthetic heme (iron-protoporphyrin IX) moiety/mole of enzyme. In spite of their identical heme moieties, P450s differ functionally, a property conferred by individual heme-apocytochrome microenvironments. P450s are instrumental in the oxidative/reductive metabolism of various physiologically relevant endobiotics and xenobiotics. However, although all these reactions result in the formation of readily excretable products, not all are beneficial. P450s catalyze the metabolism of some substrates to radicals and other reactive species that can induce toxicity/pathological damage. Furthermore, in the course of certain redox reactions, the participating P450 is sacrificed in a process classified as a mechanism-based or "suicide" inactivation. To date, three distinct mechanisms of substrate-mediated P450 inactivation have been characterized: (a) prosthetic heme destruction via N-alkyl/arylation [i.e., allylisopropylacetamide (AIA), secobarbita (SB)I; (b) apocytochrome alkylatin by a reactive intermediate (chloramphenicol, SB, 11-undecynoic acid); and (c) destruction of the prosthetic heme to products that irreversibly bind to the apocytochrome [CC14,spironolactone (SPL), 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP)I. By definition, "suicide" inactivations occur at the active site. Isolation and structural characterization of the N-alkylated heme has unequivocally established this criterion for N-alkylation of P450 heme. However, the criterion for "suicide inactivation" has not been rigorously applied to modes b or c of drug-induced P450 destruction because the inaccessibility to structural analyses of the highly hydrophobic apoP450 active site regions and their resistance to proteolytic digestion (with an array of proteases) have until now largely precluded their definitive mechanistic classification. Using lysyl endopeptidase C and/or pepsin digestion as well as CNBr cleavage, the P450 peptides modified by either the heme or the drug have been HPLC mapped and isolated as a first step to their identification and structural characterization by mass spectrometric analyses using an array of mass spectrometric techniques provided by the Mass Spectrometry Facility. To date, two different P450 peptides alkylated by heme and SB have been isolated and characterized, using ESMS and MSLDIMS. Such structural characterization will greatly contribute to the definitive mechanistic elucidation of modes b and c inactivation processes.