Purification and Properties of 4-Hydroxy-4-methyl-2-oxoglutarate Aldolase

Abstract 4-Hydroxy-4-methyl-2-oxoglutarate aldolase was purified to a state of apparent homogeneity from Pseudomonas putida. The molecular weight of the enzyme was estimated to be around 150,000 from sedimentation equilibrium and sedimentation velocity data. The physicochemical properties described include: sedimentation coefficient (s20, w0 = 7.98 S), frictional ratio (f:f0 = 1.15) and apparent partial specific volume (v = 0.742 ml g-1). The low mobility of the protein in sodium dodecyl sulfate electrophoresis greatly increased after reduction with mercaptoethanol. One main band was then observed, with the mobility expected for a protein of molecular weight about 27,000, and it appears that before reduction, six polypeptide chains of similar size were joined through disulfide linkages. In urea-acetic acid gels, electrophoresis of the enzyme before reduction revealed heterogeneity attributable to disulfide bond interchange; this may have occurred during purification. 4-Carboxy-4-hydroxy-2-oxoadipate was a substrate for the enzyme, but other hydroxyoxoacid intermediates in aromatic catabolism were not. The aldolase attacked only one enantiomer of 4-hydroxy-4-methyl-2-oxoglutarate, the reaction was inhibited competitively by oxaloacetate and the enzyme required Mn++ or Mg++ (Km = 0.17 mm MgCl2) for activity.