Dihydro-orotase

FIG. 6.12 Pyrimidine de novo synthesis pathway. Enzymes are as follows (1) carbamoyl-phosphate synthetase II (2) asparate carbamoyl-transferase (3) dihydro-orotase (4) dihydro-orotate oxidase (5) orotate phosphoribosyltransferase (6) orotidine-5 -phosphate decarboxylase (7) nucleoside monophosphate kinase (8) nucleotide diphospho kinase (9) CTP synthetase. 

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... 

All of the enzyme activities involved in de novo UMP biosynthesis have been identified in the trematodes Schistosoma mansoni (81,106,107) and S. japonicum (108). Carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydro-orotase have been partially purified from the cytosol and appear to exist, as in mammalian cells, as a multienzyme protein (81,106). Dihydro-orotate oxidase is membrane bound (81), but its electron acceptor has not been identified. Orotate PRTase and OMP decarboxylase are also cytosolic. In mammalian cells these last two enzymes exist as part of a multienzyme protein that efficiently channels orotate to UMP since little free OMP is present in the cell. In S. mansoni they also exist as a multienzyme protein but apparently the channeling is less efiBcient since levels of free OMP are significantly higher than those of UMP (107). 

Bacterial aspartate carbamoyltransferase is subject to allosteric inhibition [92] by CTP, one of the end products of the pathway. The enzyme from E. coli has been extensively studied because of its regulatory properties [92-94]. The native molecule consists of three regulatory subunits and two catalytic ones. However, three different classes of aspartate carbamoyltransferase have been recognized in different bacterial species, differing in molecular size and kinetic properties [95]. The enzyme has also been demonstrated in a number of animal tissues [96] and together with carbamoyl phosphate synthetase II and dihydro-orotase was found in... 

The enzyme catalysing the reversible cyclization of carbamoyl aspartate to dihydro-orotate is called dihydro-orotase (L-4,5-dihydro-orotate amino-hydrolase, EC 3.5.2.3). Dihydro-orotase was found in various animal tissues and for the catalytic function requires Zn + ions [98]. Orotic acid was found to be a competitive inhibitor of dihydro-orotate synthesis though a variety of other pyrimidines had no effect on enzyme activity [99]. 

Dihydro-orotate-oxidizing activity in rat liver homogenates can be recovered completely in the mitochondrial fraction [103,104].With the exception of this system all the other enzymes of the orotate pathway appear to be present in the soluble cytosolic fraction. Dihydro-orotate dehydrogenase from rat liver was found to be located on the outer surface of the inner membrane of mitochondria [105]. Dihydro-orotate can diffuse freely from the cytosol into the mitochondria and orotate can diffuse freely from the mitochondria into the cytosol. Therefore no active transport of either dihydro-orotate or orotate is required in pyrimidine synthesis [105]. In addition to inhibiting dihydro-orotase, orotic acid strongly blocks [103] dihydro-orotate oxidation. 

Five of the enzymes of UMP biosynthesis exist in the soluble fraction of Ehrlich ascites carcinoma as two enzyme complexes [143]. One complex contains the first three enzymes of the pathway, carbamoyl phosphate synthetase, aspartate carbamoyltransferase and dihydro-orotase and has an apparent molecular weight of 800000 to 850000 daltons. The second enzyme complex contains orotate phosphoribosyltransferase and orotidylic acid decarboxylase and sediments in a sucrose gradient with 30% dimethyl sulphoxide and 5% glycerol with an apparent molecular weight of 105 000 to 115000 daltons [143]. 

Glutamine-dependent carbamoyl phosphate synthetase, aspartate carbamoyltransferase and dihydro-orotase were co-purified as a high molecular weight complex from an extract of unfertilized eggs of Rana catesbeiana [144]. UTP was required to maintain the integrity of the complex during... 

Similar complexes were observed in rat livers and in other tissues [145-149]. The extensively purified complex of glutamine-dependent carbamoyl phosphate synthetase, aspartate carbamoyltransferase and dihydro-orotase from rat liver had a sedimentation coefficient of 27 S (approximately 900000 daltons). Treatment of the complex with pancreatic elastase caused a selective inactivation of carbamoyltransferase with concomitant dissociation of the complex [159]. 

First three enzymes of the pyrimidine nucleotide pathway Carbamylphosphate synthetase aspartate transcarbamylase dihydro-orotase (Coleman et al., 1977)... 

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