UMP synthase

Mutation of one of the two enzyme activities of UMP synthase leads to orotic aciduria, characterized by accumulation of its first substrate orotic acid and insufficient levels of the product UMP, which reduces availability of uridine triphosphate (UTP) and cytidine triphosphate (CTP) for use in nucleic acid synthesis. 

Orotate phosphoribosyl transferase and OMP decarboxylase are separate domains of a single polypeptide—UMP synthase. 

The end-product of pyrimidine base synthesis is orotic acid, which is converted to the nucleotide OMP by the addition of ribose 6-phosphate (donated by PRPP). OMP is then converted to UMP, which is phosphorylated to UTP. UTP is then aminated to form CTP. A deficiency of the enzyme complex (UMP synthase) that converts orotic acid to UMP causes orotic aciduria. 

The nine steps involved in the purification of UMP synthase from starting tissue are outlined in figure 6.8. All steps were carried out at 0-5°C. About 200 g of Ehrlich ascites cells, a mammalian tumor rich in the desired enzymes, was suspended in buffer and processed in a tissue homogenizer, which mechanically breaks down the tissue... 

Outline of Purification of UMP Synthase from Ehrlich s Ascites Carcinoma... 

Outline of purification scheme for UMP synthase from Ehrlich s ascites tumor cells of mice. 

The purification of two proteins, UMP synthase from mammalian tumor cells, and lactose carrier protein from E. coli bacteria is described in detail to illustrate how different fractionation methods can be combined most effectively. 

Methods for Purification and Characterization of Proteins, the primary goal is to acquaint the reader with the techniques used for protein purification. The first part of chapter 6 presents methods for protein fractionation. In the second part of this chapter, purification procedures for two proteins, UMP synthase and lactose carrier protein, are presented so that the student can see how different purification steps are combined for maximum effectiveness. 

In an inborn error of metabolism called orotic aciduria, UMP synthase is defective and the urine contains large amounts of orotate. This disorder may be treated by feeding cytidine and uridine. Large amounts of orotate may also be excreted in the urine in the various hyperammonemia syndromes. Carbamoyl phosphate, formed in the mitochondria by the action of carbamoyl phosphate synthetase I, is present in such large amounts that it spills over into the cytosol. There, the carbamoyl phosphate is rapidly converted to orotic acid and the latter is cleared by the kidneys. 

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. 

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. 

For dihydroorotate synthetase, the product of reaction 1, carbamoyl phosphate (CAP) is very unstable but is rapidly transformed by aspartate transcarbamoylase which is 50 times more active (per active site) than carbamoyl phosphate synthetase. High levels of carbamoyl aspartate (CA-asp) may be toxic, but this intermediate is rapidly consumed by the high dihydroorotase activity. Because the first three reactions are catalyzed by a single protein, the three enzyme active sites are expressed in a constant ratio under all conditions of growth this maintains CAP and CA-asp at low levels. For UMP synthase, OMP decarboxylase is far more active (per active site) than orotate PRTase, resulting in low cellular levels of the intermediate, OMP, which would otherwise be subject to enzymatic hydrolysis (in cells from higher animals). 

Figure 27-27), aspartate transcarbamoylase, and dihydroorotase activity. Each subunit of Pyr 1-3 has a molecular weight of 200,000-220,000, and the native enzyme exists as multiples of three subunits. The second gene codes for dihydroorotate dehydrogenase which is located on the outer side of the inner mitochondrial membrane. Dihydroorotate, the product of Pyr 1-3, passes freely through the outer mitochondrial membrane and converted to orotate. Orotate readily diffuses to the cytosol for conversion to UMP. The third gene codes for another multifunctional polypeptide known as UMP synthase (Pyr 5,6). Pyr 5,6 (M.W. 55,000) contains orotate phosphoribosyltransferase and orotidylate (orotidine-5 -monophosphate) decarboxylase activity. Use of multifunctional polypeptides is very efficient, since the intermediates neither accumulate nor become consumed in side reactions. They are... 

In eukaryotes, enzymes 1 -3 of Figure 22.10 are all part of a single multifunctional polypeptide chain called CAD. In mammals, reactions 5 and 6 are catalyzed by a single protein called UMP synthase. 

Hereditary orotic aciduria UMP synthase Orotic acid Growth retardation... 

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. 

The de novo pyrimidine pathway is thus compartmentalized dihydroorotate synthesized by trifunctional DHO synthetase in the cytosol passes across the outer mitochondrial membrane to be oxidized to orotate, which in turn passes back to the cytosol where it is a substrate of the bifimctional UMP synthase. 

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... 

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