The Porphobilinogen Synthase Family

描述（由申请人提供）：胆色素原合酶（PBGS）是一种古老的蛋白质，对几乎所有细胞生物体都是必需的。PBGS催化四吡咯生物合成中的第一个共同步骤（即，卟啉、叶绿素、维生素B12），其是两个分子的5-氨基乙酰丙酸缩合形成胆色素原。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Zn 2+、Mg 2+和K+之间具有独特的系统发育变异。三个假设驱动拟议的研究。首先，我们提出，有机制的PBGS之间的差异，使用Zn 2+和那些不。其次，我们提出，有一个生理意义，我们新观察到的PBGS的六聚体形式。所有先前沉积的晶体结构显示八聚体。第三，我们建议，一个亚基到亚基的通信以外的活性位点的半位点反应性明显的PBGS的结构基础。四个相互关联的目标解决这些假设。 目的1是阐明人Zn ~（2+）-PBGS催化的反应机理。一种新的方法使用了一种设计用于处理两种不同底物的变体，而不是两种ALA分子。我们建议使用动力学技术，同位素效应测定，13 C和15 N NMR，和其他合作的方法。 目的2关注新发现的PBGS的六聚体形式。六聚体-八聚体转变可能是Mg 2+激活某些PBGS变构的结构基础。我们还将探讨交替的四元形式的蛋白质和等位基因变异报告，使一些人对铅中毒的环境疾病之间的关系。 目的3描述了Mg 2+需要PBGS的调查，我们专注于机制，结构和四元结构平衡。 目的4：果蝇PBGS是研究Zn 2+利用的一个上级系统，也是研究半位点反应所需的亚基间通讯的一个很好的模型。支持这些目标的是合作探测PBGS的结构和功能，使用X射线晶体学，拉曼光谱，和分析超离心。

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.