The primary objectives of this proposed research are the understanding of the catalytic and regulatory mechanisms of selected key enzymes intimately involved in the metabolism of L-aspartate: aspartate transcarbamoylase (ATCase), aspartokinase/homoserine dehydrogenase (AK/HDH). These enzymes catalyze the following BiBi reactions: ATCase: L-Asp + carbamyl-P = N-carbamyl-L-Asp + Pi AK: L-Asp + ATP = L-Asp-P + ADP HDH: L-AspCHO + NADPH = L-Homoser + NADP+ + H+ These enzymes are regulated by inhibitors and activators. ATCase is inhibited by CTP but activated by ATP. The isozymes of AK (I and III) are inhibited by L-Thr and L-Lys respectively, but activated by monovalent cations (K+, NH4 +). Isozymes I and II of AK also contain homoserine dehydrogenase (HDH, I and II). These systems can be uniquely studied with regard to their regulatory mechanisms by newly developed methods of equilibrium isotope exchange kinetics. Computer simulation studies have proven that each of ten different modes of modifier action for any kinetic mechanism produces a uniqe set of kinetic perturbation (""""""""fingerprint"""""""") patterns. These methods allow us to identify the rate-limiting steps in turnover, define relative rate constants for catalysis and substrate association-dissociation, estimate Km and Kd values, determine kinetic mechanism on the reactant and product sides, as well as precisely distinguish the exact mode of action of each modifier studied. These approaches should resolve several crucial unanswered questions or controversies still existing with these key regulatory enzymes. (a) Do ATP and CTP simply perturb the same conformation equilibrium in opposite directions on ATCase, or do they act by separate mechanisms? Initial results , obtained before the analytical computer programs were available, suggest the latter mode. Newly isolated mutant forms of ATCase also will be used to probe this question further. (b) What are the kinetic mechanisms for AK-I and HDH-I? (c) At what steps does K+ activate AK and Thr inhibit both AK-I and HSD-I? We also propose to use equilibrium exchange kinetics to study regulation at the pathway level, using the Asp-Thr pathway enzymes of E. coli. As an adjunct method we will use NMR spectoscopy to study the divalent and monovalent metal ion sites relative to modifier and substrate sites and to each other.
