PORPHOBILINOGEN SYNTHASE FAMILY

描述：（改编自申请人的摘要）胆色素原合酶 (PBGS) 是古老的蛋白质，对几乎所有细胞生物体都是必需的。他们 催化四吡咯生物合成的第一个常见步骤（即卟啉， 叶绿素、维生素B12等），是两个分子的缩合体 5-氨基乙酰丙酸形成胆色素原。迄今为止，只有一种反应 这种复杂反应的中间体是已知的。所有 PBGS 都是金属酶 金属离子的使用在 Zn(II) 之间经历了独特的系统发育转换 和镁(II)。人类 PBGS 是铅中毒的主要目标，而较少 据报道，两个等位基因的出现频率使人类倾向于这种情况 环境病。拟议的研究涉及各个方面 PBGS 的。第一个具体目标使用新颖的“设计基因”方法来生产 并纯化 100 毫克的人类和豌豆 PBGS，因为人类 PBGS 使用 Zn(II) 和豌豆 PBGS 使用 Mg(II)。第二个目标是描述 PBGS 的特征 源自两个常见的人类等位基因并确定其分子基础 它们与 Pb(II) 相关的明显差异。这一目标包括彻底的动力 人类蛋白质的生化特征，包括 13C NMR 和 EXAFS 和拉曼光谱以及 X 射线晶体学的协作。的 拉曼数据与活性位点配体的 13C NMR 数据互补。的 第三个目标是结合动力学、金属和底物使用诱变 结合研究、磁共振技术以及协作 探索 PBGS 中结构/功能关系的技术。突变 将使我们能够表征未知的中间体 PBGS 催化反应。一些突变探测两者的个体功能 人类 PBGS 的活性位点 Zn(II) 等人探讨了 这些同八聚体蛋白质的半位点反应性。第四个目标重点 关于催化必需物质之间有趣的系统发育转换 Zn(II) 与 Mg(II)。研究针对豌豆 PBGS，并探讨了 变构 Mg(II)，在哺乳动物 PBGS 中不存在，但存在于植物和许多植物中 微生物PBGS。拟议研究的总和预计将产生新的和 有关铅中毒机制、中间体的重要信息 在 PBGS 催化反应中，Zn(II) 和 Mg(II) 的可能作用 在酶催化中，半位点反应性的结构基础，以及 PBGS 之间的系统发育差异。因为 PBGS 是一种必需酶 由于物种之间存在明显的差异，我们设想彻底的 对系统发育差异的理解将成为 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.