PORPHOBILINOGEN SYNTHASE, PROBES OF THE ACTIVE SITE

胆色素原合成酶，活性位点探针

基本信息

批准号：
3251198
负责人：
EILEEN K JAFFE
金额：
$ 25.15万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-04-01 至 1995-08-31
项目状态：
已结题

项目摘要

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.

胆色素原 (PBG) 是所有生物四吡咯的前体（例如四吡咯）。卟啉、二氢卟酚、corrin、F 430、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至关重要，并且是该计划的主要目标环境毒素铅。 PBGS 底物水平增加铅中毒者体内的 5-氨基乙酰丙酸 (ALA) 被认为会导致儿童发育迟缓，成人神经衰弱。 PBGS 是一种 Zn(II) 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 的催化机制和破译 Zn (II) 的催化和结构作用。 PBGS 催化唯一的生物不对称缩合反应 γ-酮、δ-氨基酸，但代表了更大类别的氨基酸锌金属酶和脱水酶。 PBGS 反应通过机制，其中第一个结合的 ALA 在酮碳和活性位点赖氨酸。我们已经证明 Zn(II) 和/或席夫碱的形成不需要巯基，但结合第二个 ALA 所需。使用 13C 和 15 N NMR，我们有确定 1) 酶结合的希夫碱为亚胺（而不是亚胺）烯胺）具有已知的立体化学和质子化状态，2）表明酶结合的 PBG 含有去质子化的氨基，其溶液 pKa 为 11. 核磁共振研究极大地提高了我们对核磁共振的认识 PBGS机制以及使用13C和15N NMR观察蛋白质结合配体。 PBGS 机制的其余部分仍然没有得到很好的描述，并且在相关问题中提出：1）什么是互变异构结构酶结合的 ALA？ 2) ALA 分子之间形成的第一个键是 a C-C键还是C-N键？ 3) Zn(II)的活化作用是什么以及步骤是什么是否受到铅的抑制？ 4) 功能活性位点氨基是什么酸？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，以及 NMR，以确定定义 PBGS 催化的分子结构反应。我们还将制备两种潜在中间体的类似物添加产品并将其行为描述为替代品 PBGS 的底物、可逆抑制剂或亲和标记。我们将使用内在 Zn(II) 探针以确定是否存在任何相互作用金属与基材、中间体或产品之间。作为我们化学修饰研究的补充，我们将阐明通过纯化和测序来分析 PBGS 活性位点上存在的氨基酸化学修饰的肽。