Methylmalonyl-CoA decarboxylase: Partial purification and enzymatic properties

Methylmalonyl-CoA decarboxylase, the enzyme responsible for the formation of the propionate moiety in the fermentation of lactate and the decarboxylation of succinate by Micrococcus lactilyticus, is a biotinoprotein intimately associated with the ribosomal fraction of the cell. A method for solubilizing and partially purifying this particulate enzyme is presented. The soluble enzyme which has been purified approximately 5-fold from the crude extract is free of RNA and has a molecular weight of 275,000–300,000. The decarboxylase is specific for S-methylmalonyl-CoA with an apparent K m value of 1 × 10 −6 m. At higher concentrations, the substrate is a good inhibitor of the decarboxylase and 50% inhibition is noted at 8 × 10 −5 m. The enzyme is inhibited noncompetitively by coenzyme A (K i = 4 × 10 −3 m) and by other acyl-CoA compounds, e.g. malonyl-CoA, (K i = 1 × 10 −4 m). No cofactors are required for the decarboxylation, nor do any cofactors stimulate the rate of the reaction. Net fixation of H 14CO 3 − into methylmalonyl-CoA could not be obtained even when ATP and Mg 2+ were present. An avidin sensitive exchange reaction was noted between propionyl-CoA-1- 14C and unlabeled methylmalonyl-CoA.