Publisher Summary This chapter discusses the structure and mechanism of citrate synthase. The enzyme citrate synthase that catalyzes the reaction at the entry to the citric acid cycle, forms an outstanding system for investigation of enzymatic mechanisms, enzyme–substrate interactions, conformational change, protein folding, quaternary assembly of biological structures, and organelle targeting. Citrate synthase provides a system in which the simplest example of quaternary structural assembly to an active oligomer can be studied. In eukaryotes, the enzyme is a dimer in which each subunit contributes functional groups to the active site of the other. The dimer can be reversibly denatured to inactive monomers. The enzyme requires no unusual cofactors or metal ions for activity; it is one of few enzymes that can catalyze the synthesis of a carbon–carbon bond, and it does so at a rate that approaches the diffusion limit. Citrate synthase has been discovered in essentially every organism examined. The enzyme catalyzes the reversible condensation of acetyl-CoA to form citryl-CoA, which is subsequently hydrolyzed to yield citrate and coenzyme A. Thus, the enzyme closes the loop of the citric acid cycle, regenerating the six-carbon compound citrate from the four-carbon compound oxaloacetate.