PORPHOBILINOGEN SYNTHASE, PROBES OF THE ACTIVE SITE

胆色素原（PBG）是所有生物四吡咯（例如， 卟啉、二氢卟酚、咕啉、F 430、光敏色素）。PBG合成酶（PBGS） 催化四吡咯生物合成中的第一个共同步骤。PBGS是 对所有已知的生命形式都是必不可少的，是 环境毒素铅。PBGS底物水平升高 5-氨基乙酰丙酸（ALA）在铅中毒的人被认为是导致 儿童智力迟钝和成人神经官能症。PBGS是Zn（II） 一种金属酶，其被铅抑制是金属离子的直接结果 替代。我们的目标是阐明PBGS的催化机理， 以破译Zn（II）的催化和结构作用。 PBGS催化唯一的生物不对称缩合相同的 γ-酮，δ-氨基酸，但代表较大类别的 Zn-金属酶和磷酸酶。PBGS反应通过 其中第一个结合的ALA在两个ALA之间形成席夫碱的机制。 酮碳和活性位点赖氨酸。我们已经表明，Zn（II）和/或 巯基不是席夫碱形成所必需的， 第二个ALA的绑定所需的。使用13 C和15 N NMR，我们得到 确定1）酶结合的席夫碱作为亚胺（而不是 烯胺）的已知立体化学和质子化状态和2）表明， 酶结合的PBG含有去质子化的氨基，其溶液pKa为 11.核磁共振研究大大提高了我们对 PBGS机制及利用13 C和15 N NMR观察蛋白结合 配体。 PBGS机制的其余部分仍然表征不佳， 在相关问题中提出：1）互变异构体结构是什么 酶结合的ALA吗2)ALA分子之间形成的第一个键是 C-C还是C-N键？3)Zn（II）的活化作用是什么？ 会被铅抑制吗4）氨基的功能活性部位是什么 酸？为了回答这些问题，我们结合了以下技术： 通过亲和标记、稳定同位素标记进行化学修饰， NMR，以确定限定催化的PBGS的分子结构 反应我们还将制备两种潜在中间体的类似物， 加成产物，并将其行为表征为替代 底物、可逆抑制剂或PBGS的亲和标记。我们将使用 探针的内在锌（II），以确定是否有任何相互作用 在金属和基质、中间体或产物之间。 作为我们化学修饰研究的补充，我们将阐明 存在于PBGS活性位点的氨基酸，通过纯化和测序， 化学修饰的肽

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.