Trifunctional protein :

Enzymes 7,9, and 13 form a trifunctional protein associated with the inner face of the inner mitochondrial membrane. Very-long-chain acyl-CoA dehydrogenase is also associated with other inner mitochondrial membranes while the other enzymes are in the matrix and may be loosely associated with the inner face of the inner membrane. A medium-chain 2-enoyl-CoA hydratase may also be present in the mitochondrial matrix. 

Uchicda, Y., Izai, K., Orii, T., Hashimoto, T. (1992). Novel fatty acid p-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hy-droxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein. J. Biol. Chem. 267, 1034-1041. 

Very-long-chain acyl-CoA dehydrogenase and trifunctional protein are the two inner membrane-bound... 

Matern D, Strauss AW, Hillman SL, Mayatepek E, Millington DS, Trefz FK (1999) Diagnosis of mitochondrial trifunctional protein deficiency in a blood spot from the newborn screening card by tandem mass spectrometry and DNA analysis. Pediatr Res 46 45-49... 

Hintz SR, Matern D, Strauss A, et al (2002) Early neonatal diagnosis of long-chain 3-hydroxy-acyl coenzyme a dehydrogenase and mitochondrial trifunctional protein deficiencies. Mol Genet Metab 75 120-127... 

The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the enzymes employed depending on the length of the fatty acyl chain. For fatty acyl chains of 12 or more carbons, the reactions are catalyzed by a multienzyme complex associated with the inner mitochondrial membrane, the trifunctional protein (TFP). TFP is a heterooctamer of 4/34 subunits. Each a subunit contains two activities, the enoyl-CoA hydratase and the /3-hydroxyacyl-CoA dehydrogenase the /3 subunits contain the thiolase activity. This tight association of three enzymes may allow efficient substrate channeling from one active site to the... 

K, Johnson, A.W., Bartlett, K. (2000) The mitochondrial trifunctional protein centre of a /3-oxidation metabolon Biochem. 

Kan,J. L.jJannatipour, M., Taylor, S. M., and Moran, R. G. (1993). Mouse cDNAs encoding a trifunctional protein of de novo purine synthesis and a related single-domain glyci-namide ribonucleotide synthetase. Gene, 137, 195—202. 

A second, cytosolic CPS activity (CPSII) occurs in mammals as part of the CAD trifunctional protein that catalyzes the first three steps of pyrimidine synthesis (CPSII, asparate tran-scarbamoylase, and dihydroorotase). The activities of these three enzymes—CPSII, aspartate transcarbamoylase, and dihydroorotase—result in the production of orotic acid from ammonium, bicarbonate, and ATP. CPSII has no role in ureagenesis, but orotic aciduria results from hepatocellular accumulation of carbamyl phosphate and helps distinguish CPSI deficiency from other UCDs. Defects in CPSI classically present with neonatal acute hyperammonemic encephalopathy. The plasma citrulline and urine orotic acid concentrations are both low. A definitive diagnosis can be established by enzyme assay of biopsied liver tissue or by mutation analysis. 

There are two multifunctional proteins in the pathway for de novo biosynthesis of pyrimidine nucleotides. A trifunctional protein, called dihydroorotate synthetase (or CAD, where the letters are the initials of the three enzymatic activities), catalyzes reactions 1, 2 and 3 of the pathway (HCC>5"- CAP— CA-asp—> DHO Fig. 15-15). The enzymatic activities of carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase, are contained in discrete globular domains of a single polypeptide chain of 243 kDa, where they are covalently connected by segments of polypeptide chain whch are susceptible to digestion by proteases such as trypsin. A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate— OMP—> UMP Fig. 15-15). Two enzymatic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa which associates as a dimer. 

Trifunctional Protein and Long-Chain 3-Hydroxy Acyl-CoA Dehydrogenase Deficiencies... 

Ijlst L, Ruiter JP, Hoovers JM, Jakobs ME, Wanders RJ. Common missense mutation G1528C in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Characterization and expression of the mutant protein, mutation analysis on genomic DNA and chromosomal localization of the mitochondrial trifunctional protein alpha subunit gene. J Clin Invest 1996 98 1028-33. 

Yang Z, Yamada 1, Zhao Y, et al. Prospective screening for pediatric mitochondrial trifunctional protein defects in pregnancies complicated by liver disease. JAMA 2002 288 2163-66. 

More than 20 other human genetic defects in fatty acid transport or oxidation have been documented, most much less common than the defect in MCAD. One of the most severe disorders results from loss of the long-chain /3-hydroxyacyl-CoA dehydrogenase activity of the trifunctional protein, TFP. Other disorders include defects in the a or /3 subunits that affect all three activities of TFP and cause serious heart disease and abnormal skeletal muscle. ... 

The first three reactions are catalyzed by a trifunctional protein which contains carbamoyl-phosphate synthetase II, aspartate carbamoyltransferase and dihydro-orotase. This set of reactions begins with the synthesis of carbamoyl phosphate followed by its condensation with aspartic acid. The third step involves the closure of the ring through the removal of water by the action of dihydro-orotase to yield dihydro-orotate. The fourth enzyme, dihydro-orotate oxidase, oxidizes dihydro-orotate to orotate and is a mitochondrial flavoprotein enzyme located on the outer surface of the inner membrane and utilizes NAD" " as the electron acceptor. The synthesis of UMP from orotate is catalyzed by a bifunctional protein which comprises orotate PRTase and orotidine 5 -phosphate (OMP) decarboxylase. The former phosphoribosylates orotate to give OMP the latter decarboxylates OMP to UMP, the immediate precursor for the other pyrimidine nucleotides. It is interesting to note that whereas five molecules of ATP (including the ATP used in the synthesis of PRPP) are used in the de novo synthesis of IMP, no net ATP is used in the de novo synthesis of UMP. In de novo pyrimidine synthesis, two ATP molecules are used to synthesize carbamoyl phosphate and one ATP is needed to synthesize the PRPP used by orotate PRTase but 3 ATPs... 

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