Purification and properties of malonyl-CoA synthetase from Rhizobium japonicum.
A novel malonyl-CoA synthetase was found in Rhizobium japonicum bacteroid of the soybean nodule. The levels of the enzyme in the free-living cells grown on a variety of carbon sources including glucose were similar, indicating that this enzyme is not inducible. The malonyl-CoA synthetase from glucose-grown Rhizobium japonicum was purified to homogeneity. The Mr of the enzyme was determined to be 58,000 by gel filtration on a Sephacryl S-300 and by SDS/PAGE respectively, indicating a single polypeptide enzyme. N-Terminal amino acid of the enzyme was methionine but the enzyme preparation contained about 40% de-methionylated protein. The enzyme catalyses the formation of malonyl-CoA, AMP and PPi directly from malonate, CoA and ATP in the presence of Mg2+. High substrate specificity on malonate and ATP was revealed, but Mn2+ could be substituted for Mg2+ without any difference in activity. Optimum pH was 7.9. Kinetic constants, Km and Vmax, for malonate, CoA and ATP were 200 microM and 21.3 mumol/min per mg, 87 microM and 41.7 mumol/min per mg, and 33.3 microM and 29.4 mumol/min per mg respectively. Succinate inhibited the enzyme noncompetitively, whereas AMP and ADP inhibited competitively. Diethylpyrocarbonate and pyridoxal-5'-phosphate severely inhibited the enzyme, but iodoacetamide, p-chloromercuriphenylsulphonate, N-acetylimidazole and phenylglyoxal did not.
Kim
Y S
YS
Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea.
Chae
H Z
HZ
D001426:Bacterial Proteins; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003066:Coenzyme A Ligases / Q000302:isolation & purification* / Q000378:metabolism; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D012231:Rhizobium / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D006882:Hydroxyapatites; D017886:Durapatite; D003066:Coenzyme A Ligases; C021533:malonyl-CoA synthetase