PORPHOBILINOGEN SYNTHASE FAMILY

描述：(改编自申请者摘要)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体都是必不可少的。他们 催化四吡咯生物合成的第一个常见步骤(即， 叶绿素、维生素B12等)，这是两个分子的缩合 5-氨基乙酰丙酸形成胆红素原。到目前为止，只有一个反应 中间体因这种复杂的反应而闻名。所有的PBGS都是金属酶 而金属离子的使用在锌(II)之间遭遇了独特的系统发育转换 和镁(II)。人的PBGS是铅中毒的主要靶点，而 据报道，两个等位基因的频繁出现使人类易患此病。 环境疾病。拟议的研究涉及以下各个方面： 第一个特定的目标是使用一种新的“设计师基因”方法来生产 并提纯100毫克量的人和豌豆PBGS，因为人PBGS使用 锌(II)和豌豆PBGS使用镁(II)。第二个目标是描述PBGS的特征 来源于两个常见的人类等位基因，并确定了 它们在铅(II)离子上的明显差异。这一目标包括一个彻底的动力学 和生化特性，包括~(13)C核磁共振和 合作的EXAFS和拉曼光谱以及X射线结晶学。这个 拉曼数据是对活性中心配体的~(13)C核磁共振数据的补充。这个 第三个目标将诱变与动力学、金属和底物结合起来使用 结合研究和磁共振技术，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催化的反应。一些突变探测到这两种基因各自的功能 人PBGS活性中心锌(II)等探讨其结构基础 这些同源八聚体蛋白中的半结合点反应性。第四个目标聚焦 关于催化必需基因之间耐人寻味的系统发育转换 锌(II)与镁(II)。研究针对豌豆PBGS，并探索一种 变构镁(II)，在哺乳动物的PBGS中不存在，但在植物和许多 微生物PBGS。预计拟议研究的总和将产生新的和 关于铅中毒机制的重要信息，关于中间体 在PBGS催化反应中，锌(II)和镁(II)的可能作用 在酶催化中，基于半位反应性的结构基础，以及 不同种间的系统发育差异。因为PBGS是一种必需的酶 由于物种之间的明显差异，我们设想一个彻底的 对系统发育差异的理解将构成 合理设计一类新的针对生物的药物。

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.