REGULATION OF LIVER HEME METABOLISM & CYTOCHROME P 450 INACTIVATION

肝脏血红素代谢的调节

• 批准号: 6120218
• 负责人: Maria Almira Correia
• 金额: $ 1.44万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120218
• 关键词: bioengineering /biomedical engineering; biological products; biomedical resource; enzymes; proteins; spectrometry; structural biology

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.

肝微粒体血蛋白细胞色素P450（P450） 包括多种本构和诱导酶。 这些单体 血蛋白（MW @ 50KDA）包含一个假肢血红素 （铁-核磷脂IX）酶的部分/摩尔。 尽管他们 相同的血红素部分，P450在功能上有所不同，属性 由单个血红素型色色体微环境授予。 P450 在各种的氧化/还原代谢中发挥作用 生理上相关的内生物学和异种生物。 然而， 尽管所有这些反应会导致很容易形成 排泄物的产品，并非所有人都是有益的。 P450催化 某些底物的代谢和其他反应性物种 这会引起毒性/病理损害。 此外，在 某些氧化还原反应的过程，参与的P450为 在分类为基于机制或“自杀”的过程中牺牲 失活。 迄今为止，三种不同的机制 底物介导的P450失活的表征：（a） 通过N-烷基/芳基化的假体血红素破坏[即 烯丙基丙酰胺（AIA），secobarbita（SB）I； （b）启示色 烷基蛋白通过反应性中间体（氯霉素，SB， 11-毫秒酸）; （c）破坏假肢血红素 不可逆地结合启发性色素的产品 [CC14，螺内酯（SPL）， 3,5-二甲氧基-2,6-二甲基-4-乙基-1,4-二氢吡啶（DDEP）i。 经过 定义在活动部位发生“自杀”灭活。 N-烷基化血红素的分离和结构表征 明确建立了P450 N-烷基化的标准 血红素。 但是，“自杀失活”的标准尚未 严格应用于药物诱导的P450破坏的模式B或C 因为对高度的结构分析的无法访问 疏水APOP450活动部位区域及其对 蛋白水解消化（蛋白酶阵列）到目前为止 在很大程度上排除了他们确定的机械分类。 使用 赖氨酸内肽酶C和/或胃蛋白酶消化以及CNBR 裂解，由血红素或药物修饰的P450肽 已被HPLC映射并隔离为他们的第一步 质谱法的识别和结构表征 使用由质谱技术阵列进行的分析 质谱设施。 迄今为止，两种不同的P450肽 用血红素和SB烷基化已分离和表征，使用 ESMS和MSLDIMS。 这样的结构表征将大大 有助于模式B和C的确定机械阐明 灭活过程。