The Toxicity of Methylenecyclopropylglycine: Studies of the Inhibitory Effects of (Methylenecyclopropyl)formyl-CoA on Enzymes Involved in Fatty Acid Metabolism and the Molecular Basis of Its Inactivation of Enoyl-CoA Hydratases

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Abstract

(Methylenecyclopropyl)formyl-CoA (MCPF-CoA), a toxic metabolite of methylenecyclopropylglycine (MCPG), is known to have hypoglycemic activity due to its ability to interrupt the β-oxidation pathway of fatty acid metabolism. Earlier experiments have shown that acetoacetyl-CoA thiolase, 3-ketoacyl-CoA thiolase, acyl-CoA dehydrogenases, and enoyl-CoA hydratase (ECH) are cellular targets that can be inhibited by MCPF-CoA and/or MCPG. To gain more insights with respect to the target specificity and the mode of action, we have carried out a detailed investigation of the effects of MCPF-CoA on a variety of enzymes involved in fatty acid metabolism. Our studies confirmed that MCPF-CoA is a potent inactivator for ECHs but shows little effect on other β-oxidation enzymes tested in this study. Our results also revealed that MCPF-CoA manifests distinct modes of inhibition among ECHs isolated from different sources, being a competitive inhibitor for rat liver ECH and an irreversible inactivator for the bovine liver as well as pig kidney ECH. Given the high sequence homology of the mammalian ECH genes studied so far, the structures of these proteins are expected to be similar. Thus, the effects of MCPF-CoA toward different ECHs must be governed in part by the interaction of MCPF-CoA with the active site of each ECH whose architecture may be subtly different. More importantly, the incubation results with bovine liver ECH established that MCPF-CoA inactivates this enzyme via a mechanism involving the covalent trapping of an active site nucleophile by the methylenecyclopropane ring. Since MCPF-CoA is a rare irreversible inhibitor for ECHs, it could serve as a new lead for designing more effective agents for modulating ECH activity so as to control and/or regulate fatty acid metabolism.

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