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.