Dihydroorotic acid

Very few enzymes have been characterized as lactam-forming dehydrases. One exception is carbamoylaspartic dehydrase (EC 3.5.2.3), which catalyzes the cyclization of V-carbamoylaspartic acid to dihydroorotic acid en route to pyrimidines (this enzyme is also known as dihydroorotate amidohydrolase when it catalyzes the reverse reaction). 

Figure 8.19. Sequence reactions from aspartic acid (AA) and carbamoyl phosphate (CP) to the end product, cytidine triphosphate (CTP). The first reaction is catalyzed by ATCase. The intermediary compounds are N-carbamoyl aspartic acid (N-CAA), L-dihydroorotic acid (L-DHOA), orotic acid (OA), orotidine 5 -phosphate (0-5 -P), uridine 5 -phosphate (U-5 -P), uridine diphosphate (UDP), and uridine triphosphate (UTP).

Anodic oxidation of dihydroorotic acid, its isomer, and the 1-benzyl analog of dihydroorotic acid in H20-NaOH media is an efficient synthesis of uracils (51-91%) under mild conditions 416... 

The second reaction is ring closure to form dihydroorotic acid by the enzyme dihydroorotase. This circular product contains a 6-membered ring with nitrogen and carbons located in the same positions as in the mature pyrimidine ring. 

The fourth step in the de novo synthesis of pyrimidine nucleotides—the conversion of dihydroorotic acid to orotic acid—is catalyzed by dihydroorotic acid dehydrogenase. The enzyme, located on the cytosolic side of the inner membrane of mitochondria, is a target for antitumor agents. 

The reaction mixture consisted of 0.9 mL mitochondrial suspension (0.05-4 mg protein) and 31.5 /xL 30 mM L-dihydroorotic acid. Reactions were terminated after 5 to 60 minutes by addition of 180 /xL 16% trichloroacetic acid and chilling on ice for 20 minutes. Denatured proteins were removed by centrifugation, and the supernates neutralized with alanine-Freon before analysis by HPLC. The assay was linear with protein in the range of 0.05 to 2 mg protein/mL, and with time up to at least 60 minutes. 

In the assay of Ittarat et al. (1992), dihydroorotic acid and orotic acid were detected and quantitated at 230 nm after separation on a Corasil /xBondapak Ct8 column (3.9 mm x 100 mm, 10 /xm). The mobile phase contained 3 mM PIC A (from Waters) in 5 mM ammonium dihydrogen phosphate (pH 6.0) containing 5% methanol. 

The reaction mixture consisted of 180 /xL of 180 /xM dihydroorotic acid in 10 mM Hepes-KOH buffer (pH 8.0) and 20 /xL of intact parasites. The reaction was incubated at 37°C for 25 minutes and was stopped by boiling in a water bath for 10 minutes. The mixture was centrifuged before analysis by HPLC. Blanks were performed by adding intact parasites to the reaction mixture just before boiling. Assays were linear with time and with protein up to 140 /xg. 

Dihydroorotase catalyzes the intramolecular cyclization of 7V-carbamyl-L-aspartic acid to L-dihydroorotic acid. In mammals, the activity is present in a trifunctional enzyme that catalyzes the first three steps in the de novo synthesis of pyrimidine nucleotides. 

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). 

Leflunomide (Arava) is an anti-proliferative drug that inhibits dihydroorotate dehydrogenase, an enzyme essential for the synthesis of pyrimidines such as uracil, via aromatisation of dihydroorotic acid. It also has some immunomodulatory effects and is useful for intractable cases of rheumatoid arthritis, despite being liable to cause a number of serious side-effects. It is actually a prodrug, being converted into the active compound, a nitrile formed by cleavage of the isoxazole ring (cf. 25.5.1). 

Figure 12.4-5. Metabolism of orotic acid and dihydroorotic acid 22, 24 ...

USE The l- and DL-dihydroorotic acids are precursors to the biological pyrimidines, such as thymine, uracil, cytosine and hydroxymethylcytosine. When employed in large amounts D-dihydroorotic acid is an antimetabolite of L-di-hydroorotic acid in pyrimidine utilization. It has also been found to inhibit the orotic acid utilization of those organisms... 

Leflunomide This drug inhibits dihydroorotic acid dehydrogenase, an enzyme involved in ribonucleotide synthesis. Leflunomide arrests lymphocytes in the Gj phase of the cell cycle. Leflunomide is used in rheumatoid arthritis. The drug causes alopecia, rash, and diarrhea. 

A variation on the above theme is the feeding of a slowly assimilated derivative or precursor of the growth factor to the auxotroph. For example, in E. coli, growth of a uracil auxotroph on dihydroorotic acid, instead of on uracil, gives derepressed levels of six enzymes of pyrimidine biosynthesis (Sheperdson and Pardee, 1960). 

Acylation of 5-substituted 6//-l,3,4-thiadiazines 172 with the carboxylic group of carboxysulfonamides was mediated by a mixture of A -ethyl-A -(3-dimethylamino propyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole (HOBT), and 4-methylmorpholine in DMF at 5°C to yield l,3,4-thiadiazin-2-yl-2-[(phenylsulfonyl)amino]propan-amides 173 (Equation 22) <2001JME3231>. Analogous dihydroorotic acid derivatives 174 were also prepared. Such... 

R18 Rogers, L. E. and Nicolaisen, K. Enzymatic spectropho-tometric assay for dihydroorotic acid in serum and urine. Experientia, 28, 1258-1259 (1972)... 

Z4 Zarina, L., Blugere, N. and Snikeris, D. Effect of salts of dihydroorotic acid on the structure and function of white rat liver in chronic intoxication with tetrachloromethane. Latv. PSR Zinat. Akad. Vestis, 1, 71-74 (1975) (Russ.)... 

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