P-enolpyruvate carboxykinase and pyruvate carboxylase catalyze the formation of oxaloacetate from three-carbon substrates (P-enolpyruvate or pyruvate), nucleotide triphosphates (GTP or ATP) and carbon dioxide or bicarbonate. Both reactions require one metal ion in the formation of the nucleotide metal complex and one or more additional metal ions to form catalytically competent complexes. Both enzymes are essential steps in the pathway of gluconeogenesis from lactate or alanine in mammals. The steady-state kinetic properties of these enzymes have been characterized, however many of the important features of catalysis are poorly understood. One purpose of these studies will be to characterize the interactions of substrates and metals at the catalytic sites of the enzymes. The interactions of substrates or substrate analogues with metal ions will be measured in electron paramagnetic resonance experiments which can establish direct metal substrate contacts and quantitate metal water and metal enzyme contacts. The rates of formation and dissociation of enzyme-substrate complexes will be established by substrate trapping experiments using labeled substrates. Reversibility of catalytic steps will be estimated by positional isotopic exchange experiments which can be quantitated by nuclear magnetic resonance or mass spectrometry. Heavy-atom kinetic isotope effects will be used to investigate the effects of allosteric activation on pyruvate carboxylase. These experiments will attempt to establish coordination of the nucleotide-bound metal and the enzyme-bound metals in the catalytic sites of both enzymes. Phosphobiotin or carboxyphosphate will be implicated as the intermediate in the pyruvate carboxylase reaction. The rates of catalytic steps and their reversibility will allow analysis of commitments to catalysis. These can then be used to establish the mechanism by which the allosteric activator, acetyl-CoA, activates pyruvate carboxylase and the metal activator, Mn(II), activates P-enolpyruvate carboxykinase. Heavy-atom kinetic isotope effects may be able to distinguish between changes in the transition state structure and changes in rate-limiting step(s) of pyruvate carboxylase in response to the allosteric activator.