Characterization of Aminolevulinate Synthase

四吡咯的生物合成是从简单分子开始的，只包含很少的酶促步骤。此外，四吡咯的生物合成途径在所有生命系统中都非常相似；它是由5-氨基乙酰丙酸（ALA）的生物合成引起的。在动物、真菌和一些细菌中，该途径的第一步是由ALA合酶催化的，ALA合酶是本提案的重点酶。虽然ALA合成酶自1958年被发现以来一直被认为是血红素生物合成中的关键酶，但由于无法获得高浓度纯化的活性酶，对其结构和功能的研究机会有限。该项目的长期目标是了解5-氨基乙酰丙酸是如何在高等动物中合成的，这是四吡咯生物合成途径中第一个确定的中间体，以及该过程在肝脏和红细胞中受到调节的机制。未来两年的研究计划将利用化学、生化和分子生物学方法在大肠杆菌中过表达哺乳动物5-氨基戊酸合酶基因，并以活性形式纯化过表达的酶；并鉴定了磷酸吡哆醛结合位点和5-氨基乙酰酸合酶的活性位点。由于5-氨基乙酰酸合成酶是四吡咯生物合成的第一个酶，也是肝脏中血红素生物合成途径的限速酶，因此这些研究对了解血红素、叶绿素、胆色素和胆色素的代谢具有重要意义。

The biosynthesis of tetrapyrroles is initiated from simple molecules and comprises few enzymatic steps. Moreover, the pathway for the biosynthesis of tetrapyrroles is remarkably similar in all living systems; it is initiated with the biosynthesis of 5- aminolevulinate (ALA). In animals, fungi and some bacteria, the first step of the pathway is catalyzed by ALA synthase, the enzyme of focus of this proposal. While ALA synthase has been recognized as a key enzyme in heme biosynthesis since its discovery in 1958, there has been limited opportunity to study its structure and function due to the inability to obtain high concentrations of purified, active enzyme. The long term goal of this project is to understand how 5-aminolevulinic acid, the first committed intermediate in the tetrapyrrole biosynthetic pathway, is synthesized in higher animals, and the mechanism by which this process is regulated both in liver and erythroid cells. Future studies projected for the next two years will utilize chemical, biochemical, and molecular biological approaches to overexpress the mammalian 5-aminoevulinate synthase gene in E. coli and purify the overexpressed enzyme in active form; and identify the pyridoxal phosphate binding site and active site of the 5-aminolevulinate synthase. %%% As 5-aminolevulinate synthase is the first enzyme involved in the biosynthesis of tetrapyrroles, and the rate-limiting enzyme of the heme biosynthetic pathway in the liver, these studies are of fundamental importance to the understanding of the metabolism of hemes, chlorophylls, corrins and bile pigments.