Studies on the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA: Elucidation of the inactivation mechanism and identification of cysteine-114 as the entrapped nucleophile

The inhibitory properties of (methylenecyclopropyl)formyl-CoA (MCPF-CoA), a metabolite derived from a natural amino acid, (methylenecyclopropyl)glycin e, against bovine liver enoyl-CoA hydratase (ECH) were characterized. We ha ve previously demonstrated that MCPF-CoA specifically targets ECHs, which c atalyze the reversible hydration of alpha,beta -unsaturated enoyl-CoA subst rates to the corresponding beta -hydroxyacyl-CoA products. Here, we synthes ized (R)- and (S)-diastereomers of MCPF-CoA to examine the stereoselectivit y of this inactivation. Both compounds were shown to be competent inhibitor s for bovine liver ECH with nearly identical second-order inactivation rate constants (k(inact)/K-I) and partition ratios (k(cat)/k(inact)), indicatin g that the inactivation is nonstereospecific with respect to ring cleavage. The inhibitor, upon incubation with bovine liver ECH, labels a tryptic pep tide, ALGGGXEL, near the active site of the protein, where X is the amino a cid that is covalently modified. Cloning and sequence analysis of bovine li ver ECH gene revealed the identity of the amino acid residue entrapped by M CPF-CoA as Cys-114 (mature sequence numbering). On the basis of gHMQC (grad ient heteronuclear multiple quantum coherence) analysis with [3-C-13]-label ed MCPF-CoA, the ring cleavage is most likely induced by the nucleophilic a ttack at the terminal carbon of the exomethylene group (C-2')- We propose a plausible inactivation mechanism that involves relief of ring strain and i s consistent with examples found in the literature. In addition, these stud ies provide important clues for future design of more efficient and selecti ve inhibitors to control and/or regulate fatty acid metabolism.