Mitochondrial trifunctional protein (MTP) catalyzes three consecutive step reactions in the beta-oxidation of long-chain fatty acids, and plays important roles in control and regulation of the beta-oxidation. We overexpressed in E. coli, and purified the MTP as a Mistic fusion protein, which was found to be an alpha(2)beta(2) protein complex and characterized with kinetic studies. Trimetazidine, used for treating chronic stable angina, has been proposed to be an inhibitor of the beta-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 beta-subunit inactivated by trimetazidine. The MTP beta-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 beta-subunit are positioned in the active site similarly to those of monofunctional 3-ketoacyl-CoA thiolase.