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 M(r) 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 PP(i) 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, K(m) and V(max.), for malonate, CoA and ATP were 200-mu-M and 21.3-mu-mol/min per mg, 87-mu-M and 41.7-mu-mol/min per mg, and 33.3-mu-M and 29.4-mu-mol/min per mg respectively. Succinate inhibited the enzyme non-competitively, 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.