Purification and properties of malonyl-coenzyme A decarboxylase, a regulatory enzyme from the uropygial gland of goose

Abstract

Malonyl-coenzyme A (CoA) decarboxylase (EC 4.1.1.9) was, for the first time, purified in about 33% yield from the 100,000g supernatant of the uropygial gland of the goose by means of ammonium sulfate precipitation, Sepharose 4B gel filtration, QAE-Sephadex, and carboxymethyl (CM)-Sephadex ion exchange chromatography. This enzyme constituted about 1% of the total soluble proteins of this gland, and the purified enzyme showed a specific activity of 50–80 μmol/min/mg of protein. Sedimentation velocity (7.3 S), sedimentation equilibrium, polyacrylamide disc gel electrophoresis, sodium dodecyl sulfate polyacrylamide electrophoresis, Ouchterlony double diffusion, and immunoelectrophoresis with rabbit antiserum showed that the enzyme preparation was pure. Sedimentation equilibrium experiments showed that the molecular weight of the purified enzyme was about 186,000, and sodium dodecyl sulfate electrophoresis showed only one band with a molecular weight of 47,000. Thus, malonyl-CoA decarboxylase is a tetramer of apparently identical subunits. The amino acid composition of the enzyme is reported. Dissociation of the enzyme occurred during polyacrylamide disc gel electrophoresis, but both forms of the enzyme were enzymatically active. The pH optimum of the enzyme was at about 9.5, and from the typical Michaelis-Menten kinetic data K m and V were calculated to be 0.1 m m and 85 μmol/min/mg, respectively. The decarboxylase showed a high degree of specificity for malonyl-CoA; methylmalonyl-CoA was decarboxylated at <7% of the rate at which malonyl-CoA was decarboxylated and malonic acid was not decarboxylated. No metal ion participation in decarboxylation could be detected by the addition of chelators and/or metal ions. Neither avidin alone nor in combination with biotin affected the decarboxylase activity, suggesting that biotin is not involved in the decarboxylation. Thiol-directed reagents such as p-hydroxymercuribenzoate, bromopyruvic acid, and N-ethylmaleimide severely inhibited the enzyme, whereas similar brief treatment of the enzyme with iodoacetamide showed no inhibition. MethylmalonylCoA and acetyl-CoA inhibited malonyl-CoA decarboxylase. The probable role of this enzyme in the regulation of lipid metabolism, especially branched fatty acid synthesis, is discussed.