Characterization of citrate synthase purified from Drosophila melanogaster

Citrate synthase which condenses acetyl-CoA and oxaloacetate to citrate was purified from Drosophila melanogaster. Some physicochemical as well as enzymatical properties were investigated. The optimum pH and temperature were pH 8.0-9.0 and 45 degrees C, respectively. The molecular weight of the enzyme was determined as 81,000 Da by gel filtration and the purified active enzyme consisted of two identical subunits which had a molecular mass of 48,700 on SDS-PAGE. Homogeneity of the purified enzyme was confirmed by SDS-PAGE and also by N-terminal amino acid sequence analysis. The Michaelis constants (K(m)) of the enzyme for acetyl-CoA and oxaloacetate were 6.7 microM and 3.1 microM, respectively. Kinetic studies showed that citrate synthase follows the concerted mechanism which forms a ternary complex. Propionyl-CoA, ATP, and intermediates of the TCA cycle, succinyl-CoA and alpha-ketoglutarate, behaved as inhibitors in vitro. Using pig and chicken heart enzymes for comparison, we found similarities at the N-terminal region. However, in the Ouchterlony immunodiffusion test, the polyclonal antibody raised against Drosophila citrate synthase did not show any crossreaction with pig, chicken or pigeon enzymes.