Phosphoenolpyruvate Carboxylase of Salmonella

Phosphoenolpyruvate carboxylase from Salmonella typhimurium has been purified to homogeneity. The enzyme shows a concentration-dependent aggregation in ultracentrifugal experiments. Depending upon the age of enzyme preparations the s20w values range between 11.8 to 16.9 at about 2 mg per ml and between 41.6 to 53 at concentrations higher than 5 mg per ml. In dilute solutions the enzyme has a molecular weight of 198,000 as judged by sucrose gradient centrifugations. Enzyme treated with sodium dodecyl sulfate has a molecular weight of 49,200. From amino acid composition the minimal molecular weight is calculated to be 48,980. It is concluded that the native protein is tetrameric and is probably constituted of identical subunits. There are four sulfhydryl groups per molecule of the enzyme which are exposed only on disruption of the quarternary structure of the protein by sodium dodecyl sulfate or urea. Treatment by p-mercuribenzoate results in partial inactivation of the enzyme and in the formation of several protein species with different electrophoretic mobilities. From the pattern of their migration in an electric field it is considered that they represent a series of successive oligomers in which both even and odd aggregates of a basic protomeric unit are represented. The enzyme yields cooperative rate concentration plots with P-enolpyruvate as the variable substrate. This cooperativity disappears in the presence of the activators (acetyl coenzyme A and cytidine diphosphate) of the enzyme but there are large changes in maximal velocity and Michaelis constant of the substrate. Aspartate, an inhibitor of the enzyme, produces noncompetitive inhibition. Neither the activators nor the inhibitor changes the sedimentation velocity of the enzyme.