CARBOXYLATION OF CROTONYL-CoA BY MAMMALIAN ENZYME SYSTEMS

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Abstract
In a crude dialyzed ammonium sulfate fraction (35–65% saturation) of rat liver, carbon dioxide fixation into crotonyl-CoA took place when the test system was supplemented with ATP, Mn++, glutathione, Tris–HCl buffer (pH 7.0), and KH14CO3. The products of this reaction were identified after hydrolysis as glutaconic, β-hydroxyglutaric, maionic, and 2-ethylmalonic acids. The isolation and characterization of 5-14C-glutaconyl-CoA indicated a γ-carboxylation reaction. In the presence of endogenous enoyl-CoA hydratase, crotonyl-CoA was carboxylated more readily than β-hydroxybutyryl-CoA, suggesting that the unsaturated acyl compound was the natural substrate for the enzyme system. Carboxylation of crotonyl-CoA was greatly enhanced when liver extracts were prepared from either fasted or alloxan-diabetic rats. Fixation of carbon dioxide into crotonyl-CoA was also demonstrated with an amorphous preparation of propionyl-CoA carboxylase from pig heart. The products of this reaction were identified as radioactive malonic acid and unlabeled acetaldehyde, compounds which resulted from the alkaline hydrolysis of 2-ethylidenemalonyl-CoA, formed by the α-carboxylation of crotonyl-CoA. Evidence is presented that both α- and γ-carboxylation are catalyzed by the crude liver preparation.