The long term goal of the proposed research is to understand at the molecular level how enzymes achieve their extraordinary catalytic efficiencies. To do so, we must go beyond a specification of the number and structures of stable intermediates and their rates of interconversion to an understanding of the structures of transition states and the means by which the free energy cost for reaching them has been minimized. We shall investigate three proposed origins of enzyme catalytic enhancement; local destabilization of reacting bonds in substrates, stabilization of reactive intermediates and stabilization of transition states. We have chosen two enzymes, citrate synthase and adenosine deaminase, for special emphasis because sufficient structural and kinetic data are available to suggest which catalytic strategies are used by these enzymes, and because our preliminary experiments show that intermediate structures characteristic of those strategies can be directly observed. Citrate synthase, which catalyzes the first step in the energy yielding citric acid cycle must activate both the carbonyl of a keto acid and the methyl of an acetyl thioester. One or the other of these two events is also critical to the reactions catalyzed by other quite different enyzmes that are vital to the energy yielding reactions of glycolysis and to biosynthetic steps in the synthesis of fats and cholesterol. Adenosine deaminase, whose proper functioning is necessary to the integrity of the immune response, probably operates by stabilizing a reactive intermediate, a catalytic strategy used by several other important enzymes. We shall use techniques, NMR and FTIR spectroscopy, which are sensitive to local bond distortions and to the electronic environment and thus will allow direct observation of the molecular structures which reflect the catalytic strategies being used and provide some insight into the enzyme-ligand interactions which allow them to occur. We also shall make the kinetic and equilibrium measurements which are required for proper interpretation or deeper understandng of the spectroscopic data. We shall investigate selected other systems (including malate synthase, HMG-CoA synthase, thiolase(s), cytidine and guanine deaminases) to expand the context within which we may assess the general applicability of our conclusions. A detailed understanding of metabolic processes at the most fundamental level (how enzymes work) cannot help but lead to a better understanding of disease and how to combat it. The primary thrust of this proposal is toward the answers to one of the fundamental questions in current chemical biology.