Propionic Acid Metabolism: Mechanism of the Methylmalonyl Isomerase Reaction and the Reduction of Acrylyl Coenzyme a to Propionyl Coenzyme a in Propionibacteria.

The pathways of propionate formation in the propionibacteria and of propionate oxidation in animal tissues have been well documented. In the bacteria the final step is the transfer of a carboxyl group from methylmalonyl CoAl to pyruvate with the formation of oxalacetate and propionyl CoA." I The reaction was found to involve biotin;2 the purified enzyme, methylmalonyl-oxalacetic transcarboxylase, has now been shown to contain this factor.4 Methylmalonyl CoA arises from succinyl CoA by an isomerization the mechanism of which remains obscure. The original proposal that it occurred via a transcarboxylation5 was questioned on theoretical grounds as well as by indirect experimental evidence.2 The discovery that. the reaction was dependent on the presence of the various cobamide coenzymes6 suggested that the rearrangement was analogous to the isomerization of glutamic acid to f-methylaspartic acid observed in Clostridium tetanomorphum.) In the latter case the rearrangement can be considered to be a cleavage of a propionate moiety with subsequent reattachment of the glycine residue to the a position of propionate. Using an extract of propionibacteria, Eggerer et al.8 showed that the isomerization of methylmalonyl CoA involved a similar migration of the formyl thioester group from one methylene position of the propionate moiety to the other. Similar results were obtained in our laboratory with methylmalonyl isomerase isolated from sheep kidneys, as well as from propionibacteria. Eggerer et al.8 suggested that the rearrangement of methylmalonyl CoA involved a free radical intermediate. In accord with this hypothesis, Phares et al.9 recently reported an inhibition of the reaction by chlorpromazine, an efficient electron donor. It has also been suggested10 that the reaction might involve a dehydrogenation which would weaken the bond of the formyl CoA group, permitting its translocation to the other side of the double bond. Since there has been no successful demonstration of the existence of formyl CoA, it seemed likely that the thioester would be bound to the enzyme and that free acrylic acid might then occur as a transient intermediate. This hypothesis has been tested by carrying out the isomerization of methylmalonyl CoA in the presence of isotopic acrylic acid and examining the succinate formed for radioactivity. When crude extracts of Propionibacterium shermanii were used as a source of the transcarboxylation system for the synthesis of methylmalonyl CoA, acrylic acid-C'4 was incorporated into succinate. However, subsequent study showed that the route of its incorporation was not via the methylmalonyl isomerase reaction. This was revealed by the failure to obtain incorporation of acrylate when synthetic methylmalonyl CoA was used or when the extracts were treated with ammonium sulfate before incubation. Further investigation revealed that acrylyl CoA was formed from acrylic acid,