Pyrimidine bifunctional enzyme

Five of the first six enzyme activities of pyrimidine biosynthesis reside on multifunctional polypeptides. One such polypeptide catalyzes the first three reactions of Figure 34-2 and ensures efficient channeling of carbamoyl phosphate to pyrimidine biosynthesis. A second bifunctional enzyme catalyzes reactions 5 and 6. 

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. 

In bacteria, such as E. coli, the first six reactions of the denovo pyrimidine pathway (Fig. 15-15) are catalyzed by six distinct and separable enzymes, but in higher animals reactions 1, 2 and 3 are catalyzed by a trifunctional enzyme, and reactions 5 and 6 by a bifunctional enzyme.- What advantages might there be in associations of these active sites ... 

T. cruzi. Pyrimidine metabolism in T. cruzi is very similar to that of Leishmania (Fig. 6.17). The presence of de novo pyrimidine synthesis enzymes in epimastigotes is well established (41,81,87,92). The thymidylate synthase of T. cruzi is a bifunctional protein which combines the synthase with dihydrofolate reductase as in Leishmania (91). Pyrimidine interconversion and salvage by T. cruzi epimastigotes is similar to that in Leishmania (87,90,93). 

A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate —> OMP — UMP Fig. 14-7). Two enzymic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa that associates as a dimer. 

Orotidine 5 -phosphate decarboxylase (ODCase, E. C. 4.1.1.23) catalyzes the decarboxylation of orotidine 5 -phosphate (OMP) to form uridine 5 -phos-phate in the sixth and final step of pyrimidine biosynthesis (Fig. 1) [1]. The discovery of ODCase in 1954 followed the identification, three years earlier, of orotic acid as the metabolic precursor of nucleic acids [2, 3]. ODCase is a distinct, monofunctional polypeptide in bacteria and fungi, whereas in mammals it combines with orotate phosphoribosyltransferase (OPRTase) to form the bifunctional enzyme UMP synthase. Human deficiencies in either OPRTase or ODCase activity result in an autosomal recessive disorder called hereditary orotic aciduria [4]. The disease is characterized by depleted levels of pyrimidine nucleotides in the blood and by the appearance of crystalline... 

Dihydroorotate dehydrogenase, the enzyme catalyzing the dehydrogenation of dihydroorotate to orotate (reaction 4 of the pathway Fig. 15-15), is located on the outer side of the inner mitochondrial membrane. This enzyme has FAD as a prosthetic group and in mammals electrons are passed to ubiquinone. The de novo pyrimidine pathway is thus compartmentalized dihydroorotate synthesized by trifunctional DHO synthetase in the cytosol must pass across the outer mitochondrial membrane to be oxidized to orotate, which in turn passes back to the cytosol to be a substrate for bifunctional UMP synthase. Mammalian cells contain two carbamoyl phosphate synthetases the glutamine-dependent enzyme (CPSase II) which is part of CAD, and an ammonia-dependent enzyme (CPSase /) which is found in the mitochondrial matrix, and which is used for urea and arginine biosynthesis. Under certain conditions (e.g., hyperammonemia), carbamoyl phosphate synthesized in the matrix by CPSase I may enter pyrimidine biosynthesis in the cytosol. 

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... 

S Additional information <2, 4> (<4> enzyme is probably different from previously isolated HMP kinase, because the subunit molecular masses are significantly different [4] <4> bifunctional hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase [5] <2> bifunctional hydroxy-pyrimidine kinase/thiamin-phosphate pyrophosphorylase [3]) [3-5]... 

Another form of spatial organization of metabolism that is often seen in eukaryotes but is less common in bacteria involves enzyme aggregates or multifunctional enzymes. An example is seen in S. cerevisiae where the first two reactions in pyrimidine nucleotide biosynthesis, the synthesis of carbamyl phosphate and the carbamylation of aspartate, are catalyzed by a single bifunctional protein (31). Both reactions are subject to feedback inhibition by UTP, in contrast to the situation inB. subtilis where aspartate transcarbamylase activity is not controlled. It is possible that an evolutionary advantage of the fusion of the genes... 

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