PORPHOBILINOGEN SYNTHASE, PROBES OF THE ACTIVE SITE

描述：卟啉生物合成的关键步骤是 将两个分子的氨基乙酰丙酸（ALA）缩合成 吡咯，胆色素原。该反应由以下物质催化： 卟啉原合酶（PBGS）。所有PBGS都含有至少8种金属 离子;哺乳动物酶是Zn（4）Zn（4）物种，每个物种有两个Zn（2+）。 同源八聚体结构中的二聚体单元。在每个同源二聚体中，一个Zn 结合到所谓的“A”位点，一个Zn（2+）结合在 “B”部位。该提案有四个具体目标， 确定了这种基本的结构和催化机理， 酵素第一个目的是确定化合物的化学结构。 PBGS催化的反应中间体。将采用三种方法。 首先，定点诱变将用于在 被认为起共价催化剂作用的残基，金属配体， 或布朗斯台德碱/酸。第二，将使用氟化基材 无法进行周转的。第三，快速淬火方法将是 试过了第二个目标是确定PBGS从 各种物质使用金属离子。这将涉及准备 来自不同来源的多个PBGS，具有以下各项的各种组合 Zn（2+）、Mg（2+）、Mn（2+），然后通过EXAFS分析金属连接 ESR和功能。抑制这几种形式的PBGS 第三个目标是确定Pb（2+）是否 铅抑制的机制对于所有这些酶形式是共同的。 最后完成PBGS的X射线结构测定。

DESCRIPTION: The critical step in porphyrin biosynthesis is the condensation of two molecules of amino-levulinic acid (ALA) into the monopyrrole, porphobilinogen. This reaction is catalyzed by porphorbilinogen synthase (PBGS). All PBGS contain at least 8 metal ions; the mammalian enzyme is a Zn(4)Zn(4) species, with two Zn(2+) per dimeric unit in a homooctameric structure. In each homodimer, one Zn is bound to what is known as the "A" site, one Zn(2+) is bound at the "B" site. This proposal has four specific aims focussed on determination of the structure and catalytic mechanism of this essential enzyme. The first aim is to determine the chemical structure of the PBGS-catalyzed reaction intermediates. Three approaches will be used. First, site-directed mutagenesis will be used to make substitutions at residues thought to play a role as a covalent catalyst, a metal ligand, or Bronsted base/acid. Second, fluorinated substrate(s) will be used that cannot undergo turnover. Third, rapid quench approaches will be tried. The second aim is to determine the manner by which PBGS from various species use metal ions. This will involve the preparation of a number of PBGSs from various sources, with various combinations of Zn(2+), Mg(2+), Mn(2+), followed by analysis of metal ligation by EXAFS and ESR, and function. The inhibition of these several forms of PBGS by Pb(2+) is the focus of the third aim which is to determine whether the mechanism of lead inhibition is common to all of these enzyme forms. The last aim is to complete the x-ray structural determination of PBGS.