REGULATION OF LIVER HEME METABOLISM & CYTOCHROME P 450 INACTIVATION

肝脏血红素代谢的调节

• 批准号: 6281153
• 负责人: Maria Almira Correia
• 金额: $ 1.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 1999-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6281153
• 关键词: Mammalia; animal tissue; biological products; biomedical resource; enzymes; liver; 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 hememoieties, 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 beencharacterized: (a) prosthetic heme destruction via N-alkyl/arylation [i.e., allylisopropylacetamide (AIA), secobarbita (SB)]; (b) apocytochrome alkylatin by a reactive intermediate (chloramphenicol, SB, 11-undecynoic acid); and (c) destruction of the prosthetic hemeto products that irreversibly bind to the apocytochrome [CCl4,spironolactone (SPL), 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP)]. By definition, "suicide" inactivations occur at the active site. Isolation and structural characterization of the N-alkylated hemehas 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 stepto 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 MSLDI-MS. Such structural characterization will greatly contribute to the definitive mechanistic elucidation of modes b and c inactivation processes.

肝微粒体血红素蛋白细胞色素P450（P450） 包括多种组成型和诱导型酶。 这些单体 血红素蛋白（MW@50kDa）含有一个辅血红素 （铁-原卟啉IX）部分/摩尔酶。 尽管他们 相同的血红素，P450在功能上不同， 由个体血红素-脱辅基细胞色素微环境赋予。 P450s 在各种氧化/还原代谢中起作用 生理学相关的内源性物质和外源性物质。 然而，在这方面， 尽管所有这些反应都容易形成 排泄物，并不是所有的都是有益的。 P450催化 某些底物代谢为自由基和其他活性物质 可引起毒性/病理损伤。 更以 在某些氧化还原反应过程中，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，三种不同的机制， 底物介导P450失活已经被表征：（a） 通过N-烷基化/芳基化的血红素修复破坏[即， 烯丙基异丙基乙酰胺（AIA），裂巴比妥（SB）];（B）脱辅基细胞色素 通过反应性中间体（氯霉素，SB， 11-十一碳炔酸）;和（c）人工血红素的破坏 与脱辅基细胞色素不可逆结合的产物 [CCl4、螺内酯（SPL）， 3，5-二乙氧羰基-2，6-二甲基-4-乙基-1，4-二氢吡啶（DDEP）]。 通过 定义，“自杀”失活发生在活性位点。 N-烷基化血红素的分离和结构表征 明确确立了P450 N-烷基化的标准 血红素 然而，“自杀失活”的标准还没有被确定。 严格应用于药物诱导的P450破坏的模式B或c 因为无法获得结构分析的高度 疏水性apoP 450活性位点区域及其对 蛋白水解消化（用一系列蛋白酶）迄今为止 在很大程度上排除了它们的明确的机械分类。 使用 赖氨酰内肽酶C和/或胃蛋白酶消化以及CNBr 切割，由血红素或药物修饰的P450肽 已经被HPLC映射和分离，作为他们的第一步， 质谱鉴定和结构表征 使用质谱技术阵列进行分析， 质谱仪设施。 迄今为止，两种不同的P450肽 烷基化的血红素和SB已经分离和表征，使用 ESMS和MSLDI-MS。 有助于模式B和C的确定性机理解释 灭活过程。