DESCRIPTION: (Verbatim from the Applicant's Abstract) Our long-term goal is to understand the mechanisms of enzymes that catalyze carbon-carbon bond formation via Claisen condensations. Such enzymes are prominent in biosynthetic pathways (e.g., fats and cholesterol) and in energy-yielding pathways (e.g., tricarboxylic acid and glyoxylate cycles); we concentrate on citrate synthases. Their study will advance our general understanding of enzymatic catalysis. Citrate synthases are prototypes for several important catalytic strategies: carbonyl polarization to increase reactivity of the electrophilic substrate, facilitation of formation of the nucleophilic substrate carbanion, the use of unusual ionization states of histidine residues as acid catalysts, and changes in macromolecular conformation. We have four specific goals. 1. To complete determination of the detailed reaction profile using intramolecular substrate-isotope effects and transient kinetic methods (stopped-flow fluorescence and chemical quench). Oxygen exchange will be used to detect intermediates in the hydrolysis reaction. 2. To determine the structural basis of carbonyl polarization and carbanion stabilization. Structural and computational studies have implicated previously unstudied residues. We will change these residues to ones that cannot function in the ways propose and determine the consequences for carbonyl polarization (NMR and FTIR studies) and proton transfer (exchange studies). These solution results will be correlated with X-ray structures and calculations of the effects of mutants by our collaborators. 3. To determine the ionization states of the catalytic histidines in the various complexes of the enzyme. This exceeding important (but difficult) problem will be attacked using three different methodologies, solid-state NMR, solution-state NMR, and Raman spectroscopy. 4. To investigate the catalytic role of protein dynamics and flexibility by detailed mechanistic comparisons in a series of structurally homologous citrate synthases originating in organisms optimized to function at widely different temperatures. We shall correlate the stabilities of the intermediates and transition states with temperature to reveal which elementary steps in the mechanism are modulated by protein flexibility and dynamics. We will test predictions based on previous experimental and theoretical work.