Regulation of the Aspartate Pathway of Amino Acid Biosynthesis

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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.