Mitochondrial trifunctional enzyme catalyzes the last three of the four reactions of the mitochondrial beta-oxidation pathway (PubMed:8135828, PubMed:29915090, PubMed:30850536). The mitochondrial beta-oxidation pathway is the major energy-producing process in tissues and is performed through four consecutive reactions breaking down fatty acids into acetyl-CoA (PubMed:29915090). Among the enzymes involved in this pathway, the trifunctional enzyme exhibits specificity for long-chain fatty acids (PubMed:30850536). Mitochondrial trifunctional enzyme is a heterotetrameric complex composed of two proteins, the trifunctional enzyme subunit alpha/HADHA carries the 2,3-enoyl-CoA hydratase and the 3-hydroxyacyl-CoA dehydrogenase activities, while the trifunctional enzyme subunit beta/HADHB described here bears the 3-ketoacyl-CoA thiolase activity (PubMed:8135828, PubMed:29915090, PubMed:30850536). acetyl-CoA + an acyl-CoA = a 3-oxoacyl-CoA + CoA acetyl-CoA + butanoyl-CoA = 3-oxohexanoyl-CoA + CoA acetyl-CoA + hexanoyl-CoA = 3-oxooctanoyl-CoA + CoA acetyl-CoA + octanoyl-CoA = 3-oxodecanoyl-CoA + CoA acetyl-CoA + decanoyl-CoA = 3-oxododecanoyl-CoA + CoA acetyl-CoA + dodecanoyl-CoA = 3-oxotetradecanoyl-CoA + CoA acetyl-CoA + tetradecanoyl-CoA = 3-oxohexadecanoyl-CoA + CoA Lipid metabolism; fatty acid beta-oxidation. Heterotetramer of 2 alpha/HADHA and 2 beta/HADHB subunits; forms the mitochondrial trifunctional enzyme (PubMed:29915090, PubMed:30850536). Also purified as higher order heterooligomers including a 4 alpha/HADHA and 4 beta/HADHB heterooligomer which physiological significance remains unclear (PubMed:8163672, PubMed:29915090). The mitochondrial trifunctional enzyme interacts with MTLN (PubMed:32243843). Interacts with RSAD2/viperin (PubMed:21527675). Protein stability and association with membranes require HADHA. The disease is caused by variants affecting the gene represented in this entry. Belongs to the thiolase-like superfamily. Thiolase family.