Ferrochelatase: dose porphyrin distortion control metal ion specificity?

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。螯合铁酶是最近发现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，即亚铁插入原卟啉。卟啉与铁螯合酶结合后的畸变是该酶催化机制的关键步骤。通过对螯合酶作用机理的结构和动力学数据的分析，P.I.和合作者提出，螯合酶诱导的卟啉底物畸变不仅通过降低反应活化能来提高反应速率，而且还调节了二价金属离子进入卟啉环的方式。此外，初步结果表明，仅铁螯合酶支架的少量突变就足以改变铁螯合酶对其他金属螯合酶的活性。P.I.建议使用铁螯合酶支架来评估金属离子选择性是如何在金属螯合酶家族中产生的。要解决的假设是：螯合酶，通过不同的扭曲卟啉底物，调节金属离子被纳入卟啉环。为了验证这一假设，提出以下具体目标：1。明确小鼠铁螯合酶和直接进化的具有不同金属离子特异性和酶活性的螯合酶变体诱导卟啉畸变的模式和程度。2. 评估铁螯合酶及其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. 优化进化的螯合酶变体的金属离子选择性。从集中金属螯合酶文库的构建到卟啉扭曲模式的表征，金属离子配位几何和动力学性质的实验方法的组合将使我们能够产生和分析具有细微差异的支架，但不同的金属离子选择性和螯合酶活性的变体。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。

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.