Regulation of the Aspartate Pathway of Amino Acid Biosynthesis

天冬氨酸生物合成途径导致氨基酸赖氨酸、甲硫氨酸、苏氨酸和异亮氨酸的形成。在植物和微生物中，甚至可能在高等生物中，蛋白质合成所需的氨基酸中有整整四分之一是通过这一途径连接的。该项目涉及对这一途径的调节机制的研究。这将通过催化该途径中核心反应的酶的结构和机理研究来实现，然后检查这些酶之间发生的相互作用。第一步和第三步由两种双功能同工酶催化。天冬氨酸13-半醛脱氢酶催化葡激酶和高丝氨酸脱氢酶反应之间的插入分支点反应。广泛的动力学，修饰和诱变研究将被用来检查这些酶的机制。X射线衍射研究正在进行中，以完成这一家族的酶的结构表征。动力学、色谱和交联研究将用于检查完整酶之间以及将产生和表达的分离结构域之间的关联。新组装的双功能酶将通过催化和调节结构域的交联在蛋白质水平上产生，并通过基因融合研究在遗传水平上产生。最后，在这一途径中的代谢中间体的可能通道将通过动力学研究，缔合色谱研究，化学交联和结构表征进行评估。该生物合成途径中的初始代谢中间体（酰基磷酸和半醛）在水性环境中都不稳定。保护天冬氨酸途径中的这些活性中间体不暴露于溶剂似乎有一些优势。在完成这个项目后，我们将有一个更清楚的了解蛋白质-蛋白质之间的相互作用的可溶性酶的性质和条件，有利于连续的活性位点之间的底物通道。这些问题的答案将对其他代谢途径中的酶具有普遍的应用。

The aspartate biosynthetic pathway leads to the formation of the amino acids lysine, methionine, threonine and isoleucine. In plants and microorganisms, and possibly even in higher organisms, fully one-quarter of the amino acids that are required for protein synthesis are linked through this pathway. This project involves an examination of the mechanisms of regulation of this pathway. This will be accomplished by structural and mechanistic studies of the enzymes that catalyze the core reactions in this pathway, followed by an examination of the interactions that occur between these enzymes. The first and third steps in the pathway are catalyzed by two bifunctional isoenzymes. Aspartate 13-semialdehyde dehydrogenase catalyzes the intervening branch point reaction between the aspartokinase and the homoserine dehydrogenase reactions. A wide range of kinetic, modification, and mutagenesis studies will be used to examine the mechanisms of these enzymes. X-ray diffraction studies are underway to complete the structural characterization of this family of enzymes. Kinetic, chromatographic, and cross-linking studies will be used to examine the association between the intact enzymes, and between the separated structural domains that will be produced and expressed. Newly assembled bifunctional enzymes will be created at the protein level by the cross-linking of catalytic and regulatory domains, and at the genetic level by gene fusion studies. Finally, the possible channeling of metabolic intermediates in this pathway will be assessed by kinetic studies, association chromatography studies, chemical cross-linking, and structural characterization. The initial metabolic intermediates in this biosynthetic pathway, an acyl phosphate and a semialdehyde, are both unstable in an aqueous environment. There would appear to be some advantage in protecting these reactive intermediates in the aspartate pathway from exposure to solvent. Upon completion of this project we will have a much clearer understanding of the nature of protein-protein interactions between soluble enzymes and of the conditions that favor the channeling of substrates between consecutive active sites. The answers to these questions will have general applications for enzymes in other metabolic pathways.