PORPHOBILINOGEN SYNTHASE, PROBES OF THE ACTIVE SITE

胆红素原(PBG)是所有生物四吡咯的前体。 卟啉、绿素、科林斯、F430、光敏色素)。PBG合酶(PBGS) 催化四吡咯生物合成的第一个常见步骤。PBGS是 对所有已知的生命形式都是必不可少的，也是 环境毒素铅。增加了PBGS底物的水平 铅中毒患者体内的5-氨基酮戊酸(ALA)被认为是导致 儿童发育迟缓，成人神经症。PbGS是一种锌(II) 一种金属酶，它被铅抑制是金属离子的直接结果 换人。我们的目标是阐明PBGS的催化机理和 了解锌(II)的催化和结构作用。 PBGS催化相同的唯一生物不对称缩合 伽马-酮，三角洲-氨基酸，但代表更大类别的 锌金属酶和脱水酶。PBGS反应通过一个 其中第一个要结合的Ala在 酮碳和活性中心赖氨酸。我们已经证明了锌(II)和/或 席夫碱的形成不需要巯基，但 结合第二个ALA所需的。使用13C和15N核磁共振，我们有 确定1)酶结合的席夫碱为亚胺(而不是 已知的立体化学和质子化状态的烯胺)，以及2)表明 酶结合的PBG含有去质子氨基，其溶液pKA为 11.核磁共振研究大大提高了我们对核磁共振的认识 PBGS的作用机制及用~(13)C和~(15)N核磁共振观察蛋白质结合 配基。 PBGS机制的其余部分仍然没有得到很好的描述 在相关的问题中提出：1)什么是互变异构结构 是ALA分子之间形成的第一个键 C-C或C-N键？3)锌(II)的活化作用是什么？有哪些步骤 会被铅抑制吗？4)功能活性部位氨基是什么 酸？为了回答这些问题，我们结合了以下技术 亲和标记、稳定同位素标记的化学修饰，以及 核磁共振，以确定定义PBGS催化的分子结构 反应。我们还将准备两个潜在中间体的类似物 添加产品并将其行为描述为替代产品 PBGS的底物、可逆抑制剂或亲和标记。我们将使用 探测本征锌(II)以确定是否存在任何相互作用 在金属和基材(S)、中间体或产品之间。 作为我们的化学修饰研究的补充，我们将澄清 通过纯化和测序在PBGS活性部位存在的氨基酸 化学修饰的多肽。

Porphobilinogen (PBG) is a precursor to all biological tetrapyrroles (e.g. porphyrins, chlorins, corrins, F 430, phytochromes). PBG synthase (PBGS) catalyses the first common step in tetrapyrrole biosynthesis. PBGS is essential to all known life forms and is a principle target of the environmental toxin lead. Increased levels of the PBGS substrate 5-aminolevulinate (ALA) in lead poisoned individuals is believed to cause retardation in children and neurosis in adults. PBGS is a Zn(II) metalloenzyme whose inhibition by lead is a direct consequence of metal ion substitution. Our goal is to elucidate the catalytic mechanism of PBGS and to decipher the catalytic and structural role of Zn (II). PBGS catalyzes the only biological asymmetric condensation of identical gamma-keto, delta-amino acids, but is representative of larger classes of Zn-metalloenzymes and dehydratases. The PBGS reaction proceeds via a mechanism where the first ALA to bind forms a Schiff base between the ketonic carbon and an active site lysine. We have shown that Zn(II) and/or sulfhydryl groups are not required for Schiff base formation but are required for binding of the second ALA. Using 13C and 15 N NMR, we have identified 1) the enzyme-bound Schiff base as an imine (rather than an eneamine) of known stereochemistry and protonation states and 2) shown that enzyme-bound PBG contains a deprotonated amino group whose solution pKa is 11. The NMR studies have significantly advanced both our knowledge of the PBGS mechanism and the use of 13C and 15N NMR to observe protein-bound ligands. The remainder of the PBGS mechanism remains poorly characterized and is posed in the interrelated questions: 1) What are the tautomeric structures of enzyme-bound ALA? 2) Is the first bond formed between ALA molecules a C-C or C-N bond? 3) What is the activating role of Zn(II) and what steps are inhibited by lead? and 4) What are the functional active site amino acids? To answer these questions we are combining the techniques of chemical modification by affinity labelling, stable isotope labelling, and NMR, to determine the molecular structures which define the PBGS catalyzed reaction. We will also prepare analogs of two potential intermediate addition products and characterize their behavior as alternative substrates, reversible inhibitors, or affinity labels of PBGS. We will use probes of the intrinsic Zn(II) to determine if there are any interactions between the metal and the substrate(s), intermediates, or product. Complementary to our chemical modification studies, we will elucidate the amino acids present at the PBGS active site by purifying and sequencing the chemically modified peptides.