Ferrochelatase: dose porphyrin distortion control metal ion specificity?

铁螯合酶：剂量卟啉畸变控制金属离子特异性？

• 批准号: 7367943
• 负责人: GLORIA C. FERREIRA
• 金额: $ 22.9万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367943
• 关键词: Active Sites; Address; Binding; Binding Sites; Characteristics; Data; Defect; Diagnosis; Disease; Dose; Enzymes; Erythropoiesis; Erythropoietic Protoporphyria; Evaluation; Exhibits; Facility Construction Funding Category; Family; Genes; Goals; Hematological Disease; Heme; Homeostasis; Human; Inherited; Ions; Iron; Kinetics; Lead; Libraries; Ligands; Link; Metal Ion Binding; Metals; Mitochondria; Modality; Molecular; Mus; Mutation; Parents; Pathway interactions; Patients; Photochemotherapy; Photosensitivity; Photosensitizing Agents; Physiological; Porphyrins; Prevention; Property; Proteins; Protoporphyrins; Range; Reaction; Research; Research Personnel; Site; Skin; Specificity; Substrate Specificity; Testing; Variant; base; chelation; combinatorial; cytotoxic; directed evolution; divalent metal; enzyme mechanism; falls; ferrochelatase; heme biosynthesis; improved; member; porphyrin metabolism; programs; protoporphyrin IX; reaction rate; scaffold; zinc protoporphyrin

DESCRIPTION (provided by applicant): Defects in heme biosynthesis disrupt iron homeostasis, leading to malfunction in erythropoiesis. Ferrochelatase, the only known human chelatase in the recently recognized chelatase family of enzymes, catalyzes the last step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin. Distortion of porphyrin, following its binding to ferrochelatase, is a crucial step in the catalytic mechanism of this enzyme. Analysis of structural and kinetic data pertinent to the mechanism of chelatases led the P.I. and collaborators to propose that the chelatase-induced distortion of porphyrin substrate not only enhances the reaction rate by decreasing the activation energy of the reaction but also modulates which divalent metal ion is incorporated into the porphyrin ring. In addition, preliminary results show that only a few mutations in the ferrochelatase scaffold are sufficient to alter ferrochelatase towards other metal chelatase activities. The P.I. proposes to use the ferrochelatase scaffold to assess how metal ion selectivity arises within the metal chelatase family. The hypothesis to be addressed is: Chelatases, by differentially distorting the porphyrin substrate, modulate which metal ion is incorporated into the porphyrin ring. To test this hypothesis, the following Specific Aims are proposed: 1. Define the mode and degree of porphyrin distortion induced by murine ferrochelatase and directly- evolved chelatase variants exhibiting different metal ion specificities and enzymatic activities. 2. Assess the molecular and structural determinants for metal ion selectivity and enzymatic activity of ferrochelatase and evolved variants. 3. Optimize metal ion selectivity of evolved chelatase variants. A combination of experimental approaches ranging from construction of focused metal chelatase libraries to the characterization of the mode of porphyrin distortion, metal-ion coordination geometry and kinetic properties will allow us to produce and analyze variants with subtle differences in the scaffold but different metal ion selectivities and chelatase activities. These findings will improve our understanding of heme biosynthesis and iron homeostasis and provide interpretations at a molecular level of erythropoietic disorders, and consequently a rational approach for their prevention, diagnosis and therapy.

描述（由申请人提供）：血红素生物合成缺陷破坏铁稳态，导致红细胞生成功能障碍。亚铁螯合酶是最近公认的螯合酶家族中唯一已知的人类螯合酶，它催化血红素生物合成的最后一步，即将亚铁插入原卟啉中。卟啉与亚铁螯合酶结合后发生变形，是该酶催化机制中的关键步骤。对与螯合酶机制相关的结构和动力学数据的分析导致 P.I.和合作者提出，螯合酶诱导的卟啉底物变形不仅通过降低反应活化能来提高反应速率，而且还调节掺入卟啉环的二价金属离子。此外，初步结果表明，亚铁螯合酶支架中仅少数突变就足以将亚铁螯合酶改变为其他金属螯合酶活性。 P.I.提议使用亚铁螯合酶支架来评估金属螯合酶家族中金属离子选择性如何产生。要解决的假设是：螯合酶通过差异性地扭曲卟啉底物，调节结合到卟啉环中的金属离子。为了检验这一假设，提出了以下具体目标： 1. 定义由鼠亚铁螯合酶和表现出不同金属离子特异性和酶活性的直接进化螯合酶变体诱导的卟啉畸变的模式和程度。 2. 评估亚铁螯合酶和进化变体的金属离子选择性和酶活性的分子和结构决定因素。 3. 优化进化螯合酶变体的金属离子选择性。从构建聚焦金属螯合酶库到表征卟啉变形模式、金属离子配位几何和动力学特性等实验方法的组合，将使我们能够产生和分析支架上存在细微差异但金属离子选择性不同的变体和螯合酶活性。这些发现将提高我们对血红素生物合成和铁稳态的理解，并在分子水平上解释红细胞生成障碍，从而为其预防、诊断和治疗提供合理的方法。