Argininosuccinate is a key intermediate in the metabolism of nitrogen and in the synthesis of arginine via the urea cycle. The proposed studies are aimed at determining the mechanism of action and regulatory properties of the two enzymes responsible for the synthesis and utilization of argininosuccinate from bovine liver. These two enzymes, argininosuccinate lyase and argininosuccinate synthetase, constitute the rate limiting steps in the biosynthesis of urea and arginine. Steady-state and rapid reaction kinetic studies along with magnetic resonance techniques will be the principle methods used in accomplishing these objectives. The primary aims of the proposed research are as follows: 1) Determine the role for the metal ions needed in the reaction sequence. 2) Identify the amino acids at the active site that are responsible for binding and/or catalysis. 3) Propose a mechanism for catalysis by these enzymes and determine the individual steps that control the overall reaction rates. 4) Establish the geometry of the products, reactants and allosteric modifier at the active sites. 5) Investigate the role of metabolites on the reactions of these two enzymes and their specific effects on all of the above objectives.