The Porphobilinogen Synthase Family

描述(申请人提供)：胆红素原合成酶(PBGS)是一种古老的蛋白质，几乎对所有细胞生物都是必不可少的。PBGS催化四吡咯生物合成的第一步(即，卟啉、叶绿素、维生素B12)，这是两个5-氨基乙酰丙酸分子缩合形成胆绿素原。尽管有多种晶体结构，但催化步骤的顺序和身份仍不清楚。PBGS家族由金属蛋白组成，其中金属离子的利用在锌离子、镁离子和钾离子之间具有独特的系统发育差异。有三个假设推动了这项拟议的研究。首先，我们认为在使用和不使用锌离子的PBGS之间存在机制上的差异。其次，我们认为我们新观察到的六聚体形式的PBGS具有生理意义。所有先前沉积的晶体结构都显示为八聚体。第三，我们认为活性部位外的亚基对亚单位的通讯是PBGS中明显的半部位反应性的结构基础。四个相互关联的目标解决了这些假设。 目的1旨在阐明人锌-PBGS催化反应的机理。一种新的方法使用了一种变种，旨在处理两种不同的底物，而不是两个Ala分子。我们建议使用动力学技术、同位素效应测定、13C和15N核磁共振以及其他合作方法。 目标2专注于新发现的PBGS的六聚体形式。对于某些PBGS，六聚体-八聚体转变被认为是镁离子激活变构的结构基础。我们还将探索这种蛋白质的另一种季节型与一种报告的等位基因变异之间的关系，据报道，这种变异使一些人容易患上铅中毒这一环境疾病。 目标3描述了需要PBGS的镁离子的研究，其中我们重点介绍了机理、结构和四元结构平衡。 目的4以果蝇PBGS为研究对象，它是利用PBGS研究锌离子的一个较好的系统，也是探索半结合反应所需的亚基-亚基通讯的一个很好的模型。支持这些目标的是利用X射线结晶学、拉曼光谱和分析超速离心法探测PBGS结构和功能的合作。

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.