2-methylacetoacetate reductase and possible propionyl coenzyme A condensing enzyme activity in branched chain volatile fatty acid synthesis by Ascaris lumbricoides.

The intestinal worm, Ascaris lumbricoides, ferments carbohydrate to a mixture of end products, of which the two major ones are Z-methylbutyrate and 2-methylvalerate. The mode of formation of branched chain acids with a methyl group at C-2 or other even numbered carbon atoms is still not completely understood. It has been proposed that these compounds could be formed either from the fatty acid synthetase reaction involving methylmalonyl-CoA or from propionyl-CoA via a reversal of the p oxidation pathway. Results of the present study indicate that, in Ascaris, propionyl-CoA may be the direct precursor of the branched chain volatile acids by a pathway similar to, but remarkably different from, a reverse of the /3 oxidation pathway. Neither fatty acid synthetase nor enoyl-CoA reductase activities were demonstrable in Ascaris muscle preparations by assays that successfully demonstrate these systems in other tissues. Two new enzymes have been partially purified and characterized from Ascaris mitochondria. These are the NADH-linked 2-methylacetoacetate reductase and the NADH-linked propionyl-CoA reductase (propionyl-CoA “condensing enzyme”). The condensing enzyme was assayed spectrophotometrically by coupling the product of the condensation with the 2-methylacetoacetate reductase activity. The Ascaris 2-methylacetoacetate reductase is unique in that the apparent coenzyme A ester requirement can be substituted for by the ethyl ester. Specificity studies indicated that several substrates are active as the ethyl esters, but only if they also contain a 2-methyl group. Equal reductase activities were obtained when the ethyl esters of either 2-methylacetoacetate or 2-methylpropioacetate were employed as substrates. These would be possible precursors for 2-methylbutyrate and 2-methylvalerate, respectively. Neither the lithium salts of the branched chain keto acids nor the ethyl ester of acetoacetate were acted upon. However, acetoacetyl-CoA was reduced at approximately one-third the rate of ethyl 2-methylacetoacetate. NADH is the specific