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、蛋氨酸、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种途径连接起来的。该项目涉及对这一途径的调节机制的研究。这将通过对催化该途径中核心反应的酶的结构和机制研究来完成，然后对这些酶之间发生的相互作用进行检查。该途径的第一步和第三步由两种双功能同工酶催化。天冬氨酸13-半醛脱氢酶催化天冬氨酸激酶和同丝氨酸脱氢酶反应之间的中间分支点反应。广泛的动力学、修饰和诱变研究将用于检查这些酶的机制。x射线衍射研究正在进行中，以完成该酶家族的结构表征。动力学、色谱和交联研究将用于检查完整酶之间的联系，以及将产生和表达的分离结构域之间的联系。新组装的双功能酶将在蛋白质水平上通过催化和调控结构域的交联产生，在基因水平上通过基因融合研究产生。最后，将通过动力学研究、关联色谱研究、化学交联和结构表征来评估该途径中代谢中间体的可能通道。这种生物合成途径的初始代谢中间体酰基磷酸盐和半醛在水环境中都是不稳定的。在保护天冬氨酸途径中的这些反应性中间体免受溶剂暴露方面似乎有一些优势。在完成这个项目后，我们将对可溶性酶之间蛋白质-蛋白质相互作用的性质以及有利于底物在连续活性位点之间通道的条件有更清晰的了解。这些问题的答案将对酶在其他代谢途径中的应用具有普遍意义。

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.