Ferrochelatase: dose porphyrin distortion control metal ion specificity?

描述(申请人提供)：血红素生物合成缺陷扰乱铁稳态，导致红血球生成功能障碍。铁络合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，即将亚铁插入原卟啉。卟啉与铁络合酶结合后发生扭曲，是该酶催化机理中的关键步骤。通过对有关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的扭曲不仅通过降低反应的活化能来提高反应速度，而且还调节了哪些二价金属离子被结合到了卟啉环上。此外，初步结果表明，只有铁络合酶支架中的几个突变足以将铁络合酶改变为其他金属螯合酶活性。P.I.建议使用铁络合酶支架来评估金属螯合酶家族中金属离子的选择性是如何产生的。要解决的假设是：通过不同地扭曲卟啉底物，螯合酶调节哪种金属离子被结合到卟啉环中。为了验证这一假设，提出了以下具体目标：1.确定小鼠铁络合酶和具有不同金属离子特异性和酶活性的直接进化的螯合酶变异体诱导的卟啉扭曲的方式和程度。2.对金属离子选择性和酶活性的分子和结构决定因素进行评估。3.优化进化的螯合酶变异体对金属离子的选择性。从构建聚焦金属螯合酶文库到表征卟啉扭曲模式、金属离子配位几何构型和动力学性质等实验方法的结合，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和螯合酶活性不同的变体。这些发现将提高我们对血红素生物合成和铁稳态的理解，并在分子水平上为红细胞生成障碍提供解释，从而为其预防、诊断和治疗提供合理的途径。

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.