The Porphobilinogen Synthase Family

胆色素原合酶家族

• 批准号: 7909702
• 负责人: EILEEN K JAFFE
• 金额: $ 14.75万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-06 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7909702
• 关键词: Active Sites; Address; Aminolevulinic Acid; Anabolism; Binding; Binding Sites; Biological; Characteristics; Chemicals; Chlorophyll; Clinical; Cobalamin; Collaborations; Color; Communication; Deposition; Disease; Drosophila melanogaster; Enzymes; Equilibrium; Family; Foundations; Grant; Heme; Homo; Human; Investigation; Ions; Isotopes; Kinetics; Lead; Lead Poisoning; Ligands; Magnesium; Metal Binding Site; Metalloproteins; Metals; Modeling; Motion; Organism; Phylogenetic Analysis; Physical condensation; Physiological; Pisum sativum; Plants; Porphobilinogen; Porphobilinogen Synthase; Porphyrins; Potassium; Process; Property; Protein Family; Proteins; Publications; Raman Spectrum Analysis; Reaction; Reporting; Role; Site; Structure; Structure-Activity Relationship; System; Techniques; Tetrapyrroles; Toxic Environmental Substances; Variant; Vitamin B 12; X-Ray Crystallography; Zinc; analytical ultracentrifugation; base; chemical reaction; cofactor; design; fascinate; novel; novel strategies

DESCRIPTION (provided by applicant): Porphobilinogen synthase (PBGS) is an ancient protein, essential to nearly all-cellular organisms. PBGS catalyzes the first common step in tetrapyrrole biosynthesis (i.e., porphyrin, chlorophyll, vitamin B12), which is the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. Despite multiple crystal structures, the order and identity of the catalytic steps remains unclear. The PBGS family consists of metalloproteins among which the utilization of metal ions has a unique phylogenetic variation between Zn2+, Mg2+, and K+. Three hypotheses drive the proposed studies. First, we propose that there are mechanistic differences between the PBGS that use Zn2+ and those that do not. Second, we propose that there is a physiological significance to our newly observed hexameric form of PBGS. All previously deposited crystal structures show an octamer. Third, we propose that a subunit-to-subunit communication outside the active site is the structural basis for the half-site reactivity evident in PBGS. Four interrelated Aims address these hypotheses. Aim 1 is directed at elucidating the human Zn2+-PBGS catalyzed reaction mechanism. One novel approach uses a variant designed to process two different substrates, instead of two ALA molecules. We propose to use kinetic techniques, isotope effect determinations, 13C and 15N NMR, and other collaborative approaches. Aim 2 focuses on the newly discovered hexameric form of PBGS. A hexamer-octamer transition is proposed to be the structural basis for allosteric activation by Mg 2+ for some PBGS. We will also probe for a relationship between altemate quaternary forms of the protein and an allelic variation reported to predispose some humans toward the environmental disease of lead poisoning. Aim 3 describes investigation of the Mg2+ requiring PBGS where we focus on mechanism, structure, and the quaternary structure equilibria. Aim 4 focuses on Drosophila melanogaster PBGS, which is a superior system for the study of the Zn 2+ utilizing PBGS and an excellent model for probing the subunit-to-subunit communication required for half-site reactivity. In support of these aims are collaborations to probe the structure and function of PBGS using X-ray crystallography, Raman spectroscopy, and analytical ultracentrifugation.

描述（由申请人提供）：卟啉原合酶（PBGS）是一种古老的蛋白质，对几乎全细胞生物必不可少。 PBG催化了四吡咯生物合成（即卟啉，叶绿素，维生素B12）的第一个常见步骤，即卟啉，维生素B12）是5-氨基乙酸分子的凝结以形成卟啉原。尽管有多个晶体结构，但催化步骤的顺序和身份尚不清楚。 PBGS家族由金属蛋白组成，其中金属离子的利用在Zn2+，Mg2+和K+之间具有独特的系统发育变化。三个假设推动了拟议的研究。首先，我们建议使用Zn2+的PBG与不使用的PBG之间存在机械差异。其次，我们建议我们新观察到的六聚体形式的PBG具有生理意义。所有先前沉积的晶体结构均显示八聚体。第三，我们建议在活性位点以外的亚基对亚基通信是PBG中明显的半位点反应性的结构基础。四个相互关联的目的解决了这些假设。 AIM 1致力于阐明人Zn2+-PBG催化反应机制。一种新颖的方法使用一种设计用于处理两个不同底物的变体，而不是两个ALA分子。我们建议使用动力学技术，同位素效应确定，13C和15N NMR以及其他协作方法。 AIM 2专注于新发现的六聚体形式的PBG。提出，己酰胺 - 糖浆转变是某些PBG对Mg 2+变构激活的结构基础。我们还将探测蛋白质的高度季季形式与报道的等位基因变异之间的关系，使某些人类患有铅中毒的环境疾病。 AIM 3描述了对需要PBG的MG2+的研究，我们关注机理，结构和第四纪结构平衡。 AIM 4专注于果蝇Melanogaster PBG，这是研究Zn 2+利用PBG的卓越系统，也是探测半位点反应性所需的亚基到亚基沟通的绝佳模型。为了支持这些目标，协作是使用X射线晶体学，拉曼光谱和分析性超速离心探测PBG的结构和功能。