Orotic acid nucleotide formation from

Even though orotidylic acid or orotidine was implicated in pyrimidine formation, the precise role of orotic acid per se remained to be evaluated. On the other hand, it was posable that orotic acid was a normal intermediate that condensed with a ribose compound to yield orotidine or orotidylic acid during the biosynthetic process. In support of this thesis, it was found that 5-phosphoribosyl-l-pyrophosphate was utilized for nucleotide formation from orotic acid (83). On the other hand, it was equally posable that an aliphatic compound, such as aminofumaric acid (335) or carbamylaspartic acid (339), could have coupled with a ribose compound and formed orotidine or orotidylic acid directly without the existence or participation of orotic acid per se. In this latter instance, orotic acid would not be conadered a true intermediate in pyrimidine biosynthesis but merely an accidental cleavage product of hi ly labile orotidine or orotidylic acid. At this time research in the area of purine biosynthesis indicated that a series of acyclic intermediates attached to ribose 6-phosphate were biosynthetic intermediates and that free purines per se were not (Section II, B.). 

As early as 1949, it was demonstrated that injected or " C-labeled orotic acid was readily incorporated into DNA and RNA of mammalian tissue, indicating that orotic acid is a precursor of nucleic acid pyrimidine. The next step in pyrimidine biosynthesis is the formation of the first nucleotide in the sequence. It involves the reaction between ribosyl pyrophosphate and orotic acid to yield 5 -orotidylic acid the reaction is catalyzed by orotidylic pyrophosphorylase. Thus, the first steps of pyrimidine biosynthesis differ from the early steps of purine biosynthesis in at least two ways. Orotic acid, instead of being synthesized atom by atom as is the case for the purine ring, is made from the condensation of rather large molecules, namely, carbamyl phosphate and aspartic acid. Furthermore, all the steps of purine biosynthesis occur at the level of the nucleotide, but the the pyrimidine ring is closed at the level of the base. 

If adenosine (adenine riboside) is formed by either of the two mechanisms discussed above, the formation of adenylic acid (AMP) and the pyrophosphates depends upon the introduction of a phosphate group. Kinases for the formation of riboside 5 -phosphates from the riboside and ATP are known, Another reaction sequence (reaction b) has also been found for the formation of nucleotides (IV). Individual enzymes have been found for handling orotic acid (a pyrimidine precursor) and adenine... 

Another mechanism also existed in the ascites tumor for nucleotide synthesis. This involved a direct condensation of uracil with PRPP, the compound which was an intermediate in UMP formation from orotic acid (79, 314). However, this pathway resulted in a lower synthesis of UMP and was not believed to be the major reaction for UMP formation from uracil. 

The suggestion of a control of pyrimidine synthesis stems from the observation that pyrimidine-requiring mutants of E. coli in pyrimidine-free medium accumulated carbamylaspartic acid and, to a lesser extent, dihy-droorotic acid and orotic acid this accumulation was prevented by the addition of uracil and cytosine to the medium (443). It was shown further employing enzyme preparations that cytidine and particularly cytidine 5 -phosphate were effective inhibitors of carbamylaspartic acid synthesis, suggesting that the inhibition of this enzyme by a pyrimidine nucleotide was the mechanism for the feedback control of pyrimidine biosynthesis in bacteria. The decreased formation of dihydroorotic acid and orotic acid were probably secondary events reflecting the earlier metabolic block. 

The Biosynthesis of the Pyrimidine Ring begins with aspartic acid and carbamyl phosphate. The latter is an energy-rich compound which reacts with the former to give carbamylaspartic acid. Ring closure consumes ATP and is in principle an acid amide formation (peptide synthesis). The intermediate dihydro-orotic acid is dehydrogenated to orotic acid, probably by action of a flavoprotein. Orotic acid is the key precursor of pyrimidine nucleotides. It reacts with phosphoribosyl pyrophosphate. The removal of pyrophosphate yields the nucleotide of orotic acid, whose enzymic decarboxylation produces uridine 5 -phosphate. Phosphorylation with ATP yields uridine pyrophosphate and, finally, uridine triphosphate. Beside the above pathway, there is the further possibility of converting free uracil and ribose 1-phosphate to the nucleoside and from there with ATP to the nucleotide. 

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