Characterization of mitochondrial trifunctional protein and its inactivation study for medicine development

Mitochondrial trifunctional protein (MTP) catalyzes three consecutive step reactions in the β-oxidation of long-chain fatty acids, and plays important roles in control and regulation of the β-oxidation. We overexpressed in E. coli, and purified the MTP as a Mistic fusion protein, which was found to be an α₂β₂ protein complex and characterized with kinetic studies. Trimetazidine, used for treating chronic stable angina, has been proposed to be an inhibitor of the β-subunit. We found that a catalytic cysteine residue C105 was labeled by trimetazidine through MS/MS analysis of a trimetazidine-labeled peptide fragment obtained from pepsin digested β-subunit inactivated by trimetazidine. The MTP β-subunit was then comparatively studied with monofunctional 3-ketoacyl-CoA thiolase through sequence alignment, site-directed mutagenesis, characterization of variant enzymes with kinetic studies, and homology modeling. The results indicate that the catalytic residues of the MTP β-subunit are positioned in the active site similarly to those of monofunctional 3-ketoacyl-CoA thiolase. © 2008 Elsevier B.V. All rights reserved.