Prior to the development of molecular biology physicochemical studies of enzymes were limited to studying native enzymes, proteolysed enzymes and in certain cases chemically modified enzymes. With the advent of site directed mutagenesis and bacterial expression vectors with strong promoters, it has become possible to ask questions about the role of specific amino acids in catalysis, selectively change these amino acids, produce large amounts of the mutant protein in culture, and then study the physicochemical properties of the purified protein. We have modified His274 and Asp375 in pig citrate synthase, two amino acids identified as being at the active site of the enzyme by X-ray crystallography. These amino acids were replaced not only with uncharged amino acids, but also with amino acids with different pKa's, charge and chain lengths. Kinetic analysis of these mutant enzymes along with already published crystallographic data suggest that, (1) prior to the actual condensation reaction His274 and Asp375 act in concert, maintaining the electrostatic neutrality of the His-Asp pair while facilitating the enolization of acetyl CoA without the formation of the enolate ion and (2) during the actual condensation reaction His274 accepts a proton from the hydroxide of the enol intermediate as ASP375 protonates the reaction His274 accepts a proton from the hydroxide of the enol intermediate as Asp375 protonates the thioester sulfer, forming the sulfonium ion, thus facilitating the displacement of coenzyme A by oxaloacetate.
