Pyrimidine Phosphoribosyl pyrophosphate

In many cells, the capacity for de novo synthesis to supply purines and pyrimidines is insufficient, and the salvage pathway is essential for adequate nucleotide synthesis. In patients with Lesch-Nyhan disease, an enzyme for purine salvage (hypoxanthine guanine phosphoribosyl pyrophosphate transferase, HPRT) is absent. People with this genetic deficiency have CNS deterioration, mental retardation, and spastic cerebral palsy associated with compulsive self-mutilation, Cells in the basal ganglia of the brain (fine motor control) normally have very high HPRT activity. These patients also all have hyperuricemia because purines cannot be salvaged. 

Phosphoribosyl pyrophosphate (PRPP) is important in both, and in these pathways the structure of ribose is retained in the product nucleotide, in contrast to its fate in the tryptophan and histidine biosynthetic pathways discussed earlier. An amino acid is an important precursor in each type of pathway glycine for purines and aspartate for pyrimidines. Glutamine again is the most important source of amino groups—in five different steps in the de novo pathways. Aspartate is also used as the source of an amino group in the purine pathways, in two steps. 

Pyrimidine bases are first synthesized as the free base and then converted to a nucleotide. Aspartate and carbamoyl phosphate form all components of the pyrimidine ring. Ribose 5-phosphate, which is converted to phosphoribosyl pyrophosphate (PRPP), is required to donate the sugar phosphate to form a nucleotide. The first pyrimidine nucleotide produced is orotate monophosphate (OMP). The OMP is converted to uridine monophosphate (UMP), which will become the precursor for both cytidine triphosphate (CTP) and deoxythymidine monophosphate (dTMP) production. 

Pyrimidine biosynthesis commences with a reaction between carbamyl phosphate and aspartic acid to give carbamyl aspartic acid which then nndergoes ring closure and oxidation to orotic acid. A reaction then occurs between orotic acid and 5-phosphoribosyl pyrophosphate to give orotidine-5-phosphate which on decarboxylation yields uridine-5-phosphate (UMP). By means of two successive reactions with ATP, UMP can then be converted into UTP and this by reaction with ammonia can give rise to cytidine triphosphate, CTP (11.126). 

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. 

In detail, the synthesis as studied by Buchanan and Greenberg takes the following route 5-phosphoribosylamine (stemming from phosphoribosyl pyrophosphate and glutamine, as mentioned under pyrimidines) condenses with glycine to form the amide with the aid of ATP the... 

A different, simpler , pathway is involved in the synthesis of pyrimidine nucleotides. A pyrimidine base (orotate), is synthesised first. Then the ribose is added from 5-phosphoribosyl 1-pyrophosphate. The two precursors for the formation of orotate are carbamoylphosphate and aspartate, which form carbamoyl aspartate, catalysed by aspartate carbamoyltransferase. 

The purine and pyrimidine bases can be converted to then-respective nncleotides by reaction with 5-phosphoribosyl 1-pyrophosphate. Since these bases are not very soluble, they are not transported in the blood, so that the reactions are only of qnantitative significance in the intestine, where the bases are produced by degradation of nucleotides. In contrast, in some cells, nucleosides are converted back to nucleotides by the activity of kinase enzymes. In particular, adenosine is converted to AMP, by the action of adenosine kinase, and uridine is converted to UMP by a uridine kinase... 

The atoms of the pyrimidine ring are derived from carbamoyl phosphate and aspartate, as shown in Fig. 15-14. The de novo biosynthesis of pyrimidine nucleotides is shown in Fig. 15-15. The first completely formed pyrimidine ring is that of dihydroorotate. Only after oxidation to orotate is the ribose attached to produce orotidylate. The compound 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) provides the ribose phosphate. L-Glutamine is used as a substrate donating nitrogen atoms at reactions 1 and 9, catalyzed by carbamoyl phosphate synthetase II and CTP synthetase, respectively a second... 

At this stage, orotate couples to ribose, in the form of 5-phosphoribosyl-l-pyrophosphate (PRPP), a form of ribose activated to accept nucleotide bases. PRPP is synthesized from ribose-5-phosphate, formed by the pentose phosphate pathway, by the addition of pyrophosphate from ATP. Orotate reacts with PRPP to form orotidylate, a pyrimidine nucleotide. This reaction is driven by the hydrolysis of pyrophosphate. The enzyme that catalyzes this addition, pyrimidine phosphoribosyltransferase, is homologous to a number of other phosphoribosyltransferases that add different groups to PRPP to form the other nucleotides. Orotidylate is then decarboxylated to form uridylate (IMP), a major pyrimidine nucleotide that is a precursor to RNA. This reaction is catalyzed by orotidylate decarboxylase. 

De novo purine biosynthesis, like pyrimidine biosynthesis, requires PRPP, but for purines, PRPP provides the foundation on which the bases are constructed step by step. The initial committed step is the displacement of pyrophosphate by ammonia, rather than by a preassembled base, to produce 5-phosphoribosyl-l-amine, with the amine in the P configuration. 

Phosphoribosyl-l-pyrophosphate (PRPP) is a key intermediate in nucleotide biosynthesis. It is required for de novo synthesis of purine and pyrimidine nucleotides and the salvage pathways, in which purines are converted to their respective nucleotides via transfer of ribose 1-phosphate group from PRPP to the base that is. 

The pyrimidine ring is assembled first and then linked to ribosc phosphate to form a pyrimidine nucleotide. 5-Phosphoribosyl-1-pyrophosphate is the donor of the ribose phosphate moiety. The synthesis of the pyrimidine ring starts with the formation of carbamoylas-partate from carbamoyl phosphate and aspartate, a reaction catalyzed by aspartate transcarbamoylase. Dehydration, cyclization, and oxidation yield orotate, which reacts with PRPP to give orotidylate. Decarboxylation of this pyrimidine nucleotide yields UMP. CTP is then formed by the amination of UTP,... 

However, since the fate of the PRPP can be to form purine nucleotides, pyrimidine nucleotides, or coenzymes, the control must be complex and not limited to only purine nucleotides, pyrimidine nucleotides, or coenzymes. The next enzyme under control by purine nucleotides is PRPP transamidinase, catalyzing the formation of the phosphoribosyl amide (PRPP + glutamine PRNH2 + glutamate + pyrophosphate), the first unique precursor of purines. Since the synthesis ofPRPP is slow, there must be controls to allow its utilization in other processes when purine nucleotide pools are satisfied. The control of this system is interesting and shows the phenomena of synergism. That is, AMP, the product of one branch of the pathway, can inhibit the enzyme activity approximately 10% GMP, the product of the other branch of the pathway, can also cause approximately a 10% inhibition. 

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). 

The elucidation of the last steps of pyrimidine synthesis de novo came from the study of Hurlbert and Potter [107] which showed that uridine nucleotides were intermediates in the conversion of orotate to pyrimidines of nucleic acids. UMP was the first of the three uridine 5 -phosphates to become labelled in this process [108]. The synthesis of UMP from orotate takes place in two steps the stoichiometric condensation [109] of orotic acid with 5-phosphoribosyl-l-pyrophosphate (PRPP) to form orotidine 5 -phosphate and its subsequent irreversible decarboxylation to UMP ... 

Phosphoribosyl 1-pyrophosphate. S-phos-phoribosa 1-diphosphate, PftPP an energy-rich sugar phosphate, M, 390.1, formed by transfer of a pyro-phosphoryl residue from ATP to ribose 5-phosphate. PRPP is concerned in various biosynthetic reactions, e.g. biosynthesis of purines, pyrimidines and histidine. 

Toyocamydn 4-amino-5-cyano-7-(D-ribofurano-syl)-pyrrolo-(23-d)-pyrimidine 6-amino-7-cyano-9-P-D-ribofuranosyl-7-deazapurine, a 7-deazaadenine antibiotic from Streptomyces toyocaensis and S. rimosus. M.p. 243 C. Biosynthesis is analogous to that of Tii-bericidin (see), i.e. the carbon atoms of the pyrrole ring are derived from 5-phosphoribosyl 1-pyrophosphate. T. is particularly active against Candida albicans, Saccharomyces cerevisiae and Mycobacterium tuberculosis. 

It will be shown in Chapters 8 and 12 that purines and pyrimidines may be converted to ribonucleosides by reaction with ribose-l-P, and directly to ribonucleotides by reaction with 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P). 

PP-Ribose-P is the most important ribose phosphate donor for purine metabolism (see Chapters 7, 8) and participates in several important reactions of pyrimidine metabolism (Chapters 11, 12) it also transfers this group to a number of other acceptors (Chapter 5). In the course of studies of purine ribonucleotide biosynthesis, a product of the reaction of ribose-5-P and ATP was isolated and eventually identified as 5-phosphoribosyl 1-pyrophosphate. The pyrophosphate group is in the a-configuration, is quite labile, and almost certainly reacts enzymaticaUy as the magnesium complex. It has been chemically synthesized by Tener and Khorana 33). 

Both the pyrimidines and the purines are built up from small precursor molecules which are readily available in the metabolic pool (page 185). The free bases are not synthesized as such but, while being assembled, the partially constructed ring structure reacts with a special phosphorylated pentose known as PRPP (5-phosphoribosyl-l-pyrophosphate) and forms a ribonucleotide. The deoxyribonucleotides, with the exception of TMP which is formed by methylation of deoxyuridylate, are formed by reduction of the corresponding ribonucleoside diphosphate. The conversion is precisely controlled by allosteric effects which ensure that all four deoxyribonucleotides are available in amounts appropriate for nucleic acid synthesis. 

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