Orotate 5 -monophosphate decarboxylase

The last two steps in the biosynthesis of the mononu- cl cotide uridine 5 -monophosphate (UMP) are catalyzed by (1) orotate phosphoribosyltransferase (OPRTase) and (2) orotate 5 -monophosphate decarboxylase (OMPDase). 

OMPDase = orotate 5 -monophosphate decarboxylase OPRTase = orotate phosphoribosyltransferase. b Units refer to total amount of enzyme activity. 

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... 

De novo pathway of uridine-5 -monophosphate (UMP) synthesis. Enzymes (1) carbamoyl phosphate synthetase II (2) aspartate transcarbamoylase (3) dihydroorotase (4) dihydroorotate dehydrogenase (5) orotate phosphoribosyltransferase (6) orotidine-5 -monophosphate decarboxylase (orotidylate decarboxyla.se). 

Schematic representation of the intracellular location of the six enzymes of UMP biosynthesis in animals. Pyr 1-3 = 1, Carbamoyl phosphate synthetase II 2, aspartate transcarbamoylase 3, dihydroorotase 4, dihydroorotate dehydrogenase Pyr 5,6 = 5, orotate phosphoribosyltransferase 6, orotidine-5 -monophosphate decarboxylase.

Krungkrai, S. R., DelFraino, B. J., Smiley, J. A., Prapunwattana, P., Mitamura, T., Horii, T., and Krungkrai, J. (2005). A novel enzyme complex of orotate phosphoribosyltransferase and orotidine 5 -monophosphate decarboxylase in human malaria parasite Plasmodium falciparum Physical association, kinetics, and inhibition characterization. Biochemistry 44, 1643-1652. 

K56 Krooth, R. S., Lam, G. F. M. and Chen Kiang, S. Y. Oxipurinol and orotic aciduria effect on the orotidine-5 -monophosphate decarboxylase activity of cultured human fibroblasts. Cell, 3, 55-57 (1974)... 

The number of inherited defects of the pyrimidine metabolism described so far is small, compared to that of the purine metabolism. Combined deficiency of orotate phosphoribosyltransferase (OPRT) (EC 2.4.2.10) and orotidine 5 -monophosphate decarboxylase (ODC) (EC 4.1.1.23), designated as type I hereditary orotic aciduria, presents with characteristic clinical features such as hypochromic anemia with a megaloblastic bone marrow and crystalluria. Only six patients have been described and, as far as we know, new cases have not been discovered recently. ODC deficiency with similar clinical phenomena and leading to increased urinary excretion of orotate and orotidine has been detected in only one patient (1). A third defect, a deficiency of pyrimidine 5 -nucleotidase (Py-5NX (EC 3.1.3.5.) in erythrocytes, is associated with chronic hemolytic anemia and prominent basophylic stippling of the erythrocytes due to accumulated pyrimidine nucleotides. An increasing number of patients have been reported, their detection being facilitated by the typical phenomena. We do not know whether the urinary pyrimidine profile in this condition is abnormal. 

The process of substitution undertaken on carboxylic acids and the derivatives of carboxylic acids (anhydrides, acid halides, esters, amides, and nitriles) generally involves a series of replacement processes. Thus, individually, substitution may involve replacement of (a) the proton attached to oxygen of the -OH group (i.e., ionization of the acid) (b) the hydroxyl (-OH) portion of the carboxylic acid (or derivative) (e.g., esterification) (c) the carbonyl oxygen and the hydroxyl (-OH) (e.g., orthoester formation, vide infra) (d) the entire carboxylic acid functionality (e.g., the Hunsdiecker reaction, already discussed Scheme 9.101) and the decarboxylation of orotic acid (as orotidine monophosphate) to uracil (as uridine monophosphate)—catalyzed by the enzyme orotidine monophosphate decarboxylase (Scheme 9.115) or (e) the protons (if any) on the carbon to which the carboxylic acid functional group is attached (e.g., the Dieckman cycUzation, already discussed earlier, c Equation 9.91). Indeed, processes already discussed (i.e., reduction and oxidation) have also accomplished some of these ends. Some additional substitutions for the carboxylic acid group itself are presented in Table 9.6, while other substitutions for derivatives of carboxylic acids are shown in Tables 9.7-9.10 and discussed subsequently. 

In hereditary orotic aciduria, orotic acid is excreted in the urine because the enzymes that convert it to uridine monophosphate, orotate phosphoribosyl transferase and orotidine 5 -phosphate decarboxylase, are defective (see Figure 7-20). Pyrimidines cannot be synthesized, and therefore, normal growth does not occur. Oral administration of uridine bypasses the metabolic block and provides the body with a source of pyrimidines. 

Reaction of aspartic acid (14) with carbamoyl phosphoric acid (17) in the presence of the allosteric enzyme aspartate carbamoyltransferase (aspartate transcar-bamoylase) gives N-carbamoyl aspartic acid (18), which is cyclised to L-dihy-droorotic acid (19) by dihydroorotase. Oxidation of L-dihydroorotic acid by flavoprotein, orotate reductase gives orotic acid (20), which reacts with 5-phosphori-bosy 1-1-pyrophosphate (PRPP) in the presence of orotate phosphoribosyl transferase to form orotidine 5 -monophosphate (OMP, 21). Decarboxylation of OMP by orotid-ine 5 -phosphate decarboxylase yields uridine 5 -monophosphate (UMP, 22), which acts as precursor for the cytidine nucleotides (CTP) (Chart 6). 

The reaction of carbamoyl phosphate with aspartate to produce W-carbamo-ylaspartate is the committed step in pyrimidine biosynthesis. The compounds involved in reactions up to this point in the pathway can play other roles in metabolism after this point, A -carbamoylaspartate can be used only to produce pyrimidines—thus the term committed step. This reaction is catalyzed by aspartate transcarbamoylase, which we discussed in detail in Ghapter 7 as a prime example of an allosteric enzyme subject to feedback regulation. The next step, the conversion of A-carbamoylaspartate to dihydroorotate, takes place in a reaction that involves an intramolecular dehydration (loss of water) as well as cyclization. This reaction is catalyzed by dihydroorotase. Dihydroorotate is converted to orotate by dihydroorotate dehydrogenase, with the concomitant conversion of NAD to NADH. A pyrimidine nucleotide is now formed by the reaction of orotate with PRPP to give orotidine-5 -monophosphate (OMP), which is a reaction similar to the one that takes place in purine salvage (Section 23.8). Orotate phosphoribosyltransferase catalyzes this reaction. Finally, orotidine-5 -phosphate decarboxylase catalyzes the conversion of OMP to UMP... 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS), and the bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP (adenine dinucleotide phosphate). Ammonia then reacts with carboxyphosphate to form carbamic acid. The latter is phosphorylated by another molecule of ATP with the mediation of CPS to form carbamoyl phosphate, which reacts with aspartate by aspartate transcarbamoy-lase to form A-carbamoylaspartate. The latter cyclizes to form dihydroorotate, which is then oxidized by NAD-1- to generate orotate. Reaction of orotate with 5-phosphoribosyl-l-pyrophosphate (PRPP), catalyzed by pyrimidine PT, forms the pyrimidine nucleotide orotidylate. This reaction is driven by the hydrolysis of pyrophosphate. Decarboxylatin of orotidylate, catalyzed by orotidylate decarboxylase, forms uridylate (uridine-5 -monophosphate, UMP), a major pyrimidine nucleotide that is a precursor of RNA (Figure 6.53). 

Orotidyhc acid decarboxylase (orotidine 5 -phosphate carboxy-lyase, EC 4.1.1.23) catalyses the only irreversible step in the pyrimidine synthesis de novo. The enzyme is competitively inhibited by UMP and CMP [114-116] and some anomalous pyrimidine nucleoside 5 -monophosphates. The activity of orotidylic acid decarboxylase in excess of that of orotate phosphoribosyltransferase accounts for the absence of orotidine 5 -phosphate in the pool of low molecular weight compounds in animal cells. 

The first known drug affecting the orotate pathway was 6-azauridine [245,246], This analogue is phosphorylated to 6-azauridine 5 -monophosphate which acts as a competitive inhibitor of orotidylic acid decarboxylase [247], Therapeutic use of 6-azauridine [248,249] is occasionally complicated by a pronounced crystalluria. Owing to the block of orotidylic acid decarboxylase, large amounts of orotidine and orotic acid are excreted in urine. After the infusion of 6-azauridine the excretion of orotic acid precedes orotidine and the former disappears more rapidly from the urine. Psoriatic patients on azaribine (triacetylated form of 6-azauridine given orally) excreted 0.2-1.3 g of orotic acid and orotidine per day [250]. 

Aspartate carbamyltransferase 2 dihydroorotase 3 orotate reductase 4 orotate phosphoribosyl-transferase 5 orotidine-5 -phosphate decarboxylase 6 cytidylate kinase, nucleotide diphosphate kinase 7 cytidine triphosphate synthetase 8 nucleoside monophosphate kinase, ribonucleoside diphosphate reductase, phosphatase 9 thymidylate synthase... 

This is in agreement with our results obtained in human cultured lymphoblasts. Oxipurinol inhibits orotidyl decarboxylase(ODC), Purines might inhibit the conversion of orotic acid to orotidine monophosphate by lowering the intracellular PRPP-concentration (Crandall et al. 1978). 

The final steps of pyrimidine biosynthesis novo which are catalyzed by two sequential enzymes, orotate phosphoribosyltransfer-ase (OPRT) and orotidylic decarboxylase (ODC), involve the PP-ribose P dependent conversion of orotic acid to orotidine-5 -monophosphate (OMP) followed by decarboxylation at the 7 position to form uridine 5 -monophosphate (UMP) (Fig. 1). UMP is then utilized further in the synthesis of nucleic acids and co-enzymes. Defects at this site in this metabolic pathway are important for they can result in "pyrimidine starvation" from depletion of the intracellular pool of pyrimidine nucleotides. In man the rare genetic disease, orotic aciduria, involves a deficiency of both OPRT and ODC (Type 1) (Smith, Sullivan and Huguley, 1961) or, less commonly, only ODC (Type II) (Fox, 0 Sullivan and Firken, 1969). 

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