A revised mechanism for the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA based on unexpected results with the C114A mutant.
The compound (methylenecyclopropyl)formyl-CoA (MCPF-CoA) has been reported earlier as a potent active site-directed inactivator of bovine liver enoyl-CoA hydratase (ECH). It is believed that the mechanism of inactivation involves the attack of Cys114 at C-2' of MCPF-CoA, resulting in ring cleavage and permanent covalent modification of the enzyme. Here, we describe studies with the C114A mutant of bovine liver ECH, which was constructed and purified to determine the role of this residue in the catalytic mechanism of the enzyme. The C114A mutant, which is catalytically competent, shows an unexpected susceptibility to inactivation by MCPF-CoA, indicating that Cys114 is not the primary nucleophile responsible for the inactivation of the enzyme. To determine if catalytic residues Glu115 and Glu135 play a role in the inactivation of the enzyme, the E115Q and E135Q mutants were also constructed and purified. It was determined that these mutants did not react with MCPF-CoA, indicating a possible role for both residues in the inactivation of the wild-type enzyme. Pepsin digestion and subsequent LC-MS/MS analysis of the inactivated wild-type enzyme and C114A mutant revealed that Glu115 was modified in each case, supporting the hypothesis that this residue is the true nucleophile that traps MCPF-CoA and indicating that the covalent modification of Cys114 reported earlier may be a postinactivation artifact. We propose a modified mechanism of inactivation involving Glu115 and Glu135, and suggest that MCPF-CoA may be a mechanism-based inhibitor for bovine liver ECH.
Agnihotri
Gautam
G
Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
He
Shouming
S
Hong
Lin
L
Dakoji
Srikanth
S
Withers
Stephen G
SG
Liu
Hung-wen
HW
D000214:Acyl Coenzyme A / Q000494:pharmacology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000235:genetics; D004746:Enoyl-CoA Hydratase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
C439085:(methylenecyclopropyl)formyl coenzyme A; D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; D004247:DNA; D004746:Enoyl-CoA Hydratase