Malonyl coenzyme A synthetase. Purification and properties.
Malonyl coenzyme A synthetase (EC 6.2.1.14) was induced in Pseudomonas fluorescens grown on malonate as a sole carbon source. This enzyme was purified, for the first time, over 30-fold by the combination of ammonium sulfate precipitation, Sephadex G-150 gel filtration, DEAE-Sephacel ion exchange chromatography, and hydroxylapatite chromatography. The purified enzyme, which had a specific activity of about 0.512 mumol/min/mg, appeared to be electrophoretically homogeneous. The molecular size of the enzyme was determined to be 98,000 Da which is composed of two 49,000-Da subunits. The optimum pH for the enzyme was 7.5. Malonyl coenzyme A synthetase requires ATP, CoA, and Mg2+ for the full enzyme activity. With succinate or acetate, the synthetic rate of CoA derivative was 40% of that observed with malonate. The malonyl coenzyme A synthetase showed typical Michaelis-Menten kinetics for the substrate, malonate, ATP, and coenzyme A, from which the Km values were calculated to be 3.8 X 10(-4) M, 2 X 10(-3) M, and 10(-4) M and Vmax values to be 0.117 mumol/min/mg, 0.111 mumol/min/mg, and 0.142 mumol/min/mg, respectively. The purified malonyl coenzyme A synthetase was immunogenic in the rabbit and Ouchterlony double diffusion analysis revealed a single precipitant line with the enzyme. The antiserum inhibited the enzyme activity and the extent of inhibition was dependent on the amount of the serum added.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000302:isolation & purification*; D004589:Electrophoresis, Disc; D005779:Immunodiffusion; D008274:Magnesium / Q000378:metabolism; D008314:Malonates / Q000378:metabolism; D008970:Molecular Weight; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008314:Malonates; D000255:Adenosine Triphosphate; C030290:malonic acid; D003066:Coenzyme A Ligases; C021533:malonyl-CoA synthetase; D008274:Magnesium; D003065:Coenzyme A