PORPHOBILINOGEN SYNTHASE FAMILY

描述：（改编自申请人的摘要）胆色素原胆色素酶 (PBGS的）是古老的蛋白质，对几乎所有的细胞有机体都是必需的。他们 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等），这是两个分子的凝聚， 5-氨基乙酰丙酸形成胆色素原。迄今为止，只有一种反应 已知这种复杂反应的中间体。所有PBGS都是金属酶 和金属离子的使用遭受了一个独特的系统发育开关之间的锌（II） 和Mg（II）。人体PBGS是铅中毒的主要靶点， 据报道，两个等位基因的频率使人类倾向于这种情况。 环境病。拟议的研究涉及 PBGS的。第一个特定的目标使用一种新的“设计基因”方法来产生 并纯化100 mg量的人和豌豆PBGS，因为人PBGS使用 Zn（II）和豌豆PBGS使用Mg（II）。第二个目的是表征PBGS 来自两个常见的人类等位基因，并确定了 与Pb（II）有关的明显差异。这一目标包括彻底的动力学 和人类蛋白质的生物化学表征，包括13 C NMR和 合作EXAFS和拉曼光谱和X射线晶体学。的 拉曼数据与活性位点配体上的13 C NMR数据互补。的 第三个目的是结合动力学、金属和底物使用诱变 结合研究和磁共振技术，加上合作 技术来探测PBGS中的结构/功能关系。突变 将使我们能够描述未知的中间体， PBGS催化反应。一些突变会探测这两种基因的个别功能 人PBGS的活性位点Zn（II）和其他探针的结构基础 这些同源八聚体蛋白中的半位点反应性。第四个目标是 在催化必需的 Zn（II）与Mg（II）。研究针对豌豆PBGS，并探讨了一种新的方法。 别构Mg（II），在哺乳动物PBGS中不存在，但存在于植物和许多 微生物PBGS。拟议研究的总和预计将产生新的和 关于铅中毒机制的重要信息， 在PBGS催化的反应中，Zn（II）和Mg（II）的可能作用 在酶催化中，基于半位点反应性的结构基础， PBGS之间的系统发育差异。因为PBGS是一种必需的酶 由于物种之间存在明显的差异，我们设想一个彻底的 对系统发育差异的理解将构成 合理设计一类新的生物体特异性药物。

DESCRIPTION: (Adapted from Applicant's Abstract) The porphobilinogen synthases (PBGS's) are ancient proteins, essential to nearly all cellular organisms. They catalyze the first common step in tetrapyrrole biosynthesis (i.e., porphyrin, chlorophyll, vitamin B12, etc.), which is the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. To date, only one reaction intermediate is known for this complex reaction. All PBGS's are metalloenzymes and metal ion usage has suffered a unique phylogenetic switch between Zn(II) and Mg(II). Human PBGS is the primary target for lead poisoning and the less frequent of two alleles is reported to predispose humans toward this environmental disease. The proposed studies address various aspects of the PBGS's. The first specific aim uses a novel "designer gene" approach to produce and purify 100 mg quantities of human and pea PBGS because human PBGS uses Zn(II) and pea PBGS uses Mg(II). The second aim is to characterize the PBGS derived from two common human alleles and determine the molecular basis for their apparent Pb(II)-related differences. This aim includes a thorough kinetic and biochemical characterization of the human proteins, including 13C NMR and collaborative EXAFS and Raman spectroscopies and X-ray crystallography. The Raman data are complementary to the 13C NMR data on active site ligands. The third aim uses mutagenesis in conjunction with kinetics, metal and substrate binding studies, and magnetic resonance techniques, plus collaborative techniques to probe structure/function relationships in PBGS. The mutations will allow us to characterize the yet unknown intermediates in the PBGS-catalyzed reaction. Some mutations probe individual functions of the two active site Zn(II) of human PBGS and others probe the structural basis for half-sites reactivity in these homo-octameric proteins. The fourth aim focuses on the intriguing phylogenetic switch between the catalytically essential Zn(II) vs. Mg(II). Studies are directed at pea PBGS and also probe an allosteric Mg(II), absent in mammalian PBGS but present in plant and many microbial PBGS. The sum of the proposed studies is expected to yield new and important information on the mechanism of lead poisoning, on the intermediate in the PBGS catalyzed reaction, on the possible roles of both Zn(II) and Mg(II) in enzyme catalysis, on the structural basis for half sites reactivity, and on the phylogenetic differences between PBGSs. Because PBGS is an essential enzyme with distinct variations between species, we envision that a thorough understanding of the phylogenetic differences will form the basis for the rational design of a new class of organism-specific drugs.