INTRODUCTION – BIOSYNTHESIS OF NUCLEOTIDE
Nucleotides have a variety of important functions in all cells. They are the precursors of DNA and RNA. They are essential carriers of chemical energy—a role primarily of ATP and to some extent GTP. They are components of the cofactors NAD, FAD, Sadenosylmethionine, and coenzyme A, as well as of activated biosynthetic intermediates such as UDP-glucose and CDP-diacylglycerol. Some, such as cAMP and cGMP, are also cellular second messengers
Two types of pathways lead to nucleotides: the de novo pathways and the salvage pathways. De novo synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose 5-phosphate, CO2 , and NH3 . Salvage pathways recycle the free bases and nucleosides released from nucleic acid breakdown. Both types of pathways are important in cellular metabolism and both are discussed in this section.
The de novo pathways for purine and pyrimidine biosynthesis seem to be nearly identical in all living organisms. Notably, the free bases guanine, adenine, thymine, cytidine, and uracil are not intermediates in these pathways; that is, the bases are not synthesized and then attached to ribose, as might be expected.
The purine ring structure is built up one or a few atoms at a time, attached to ribose throughout the process. The pyrimidine ring is synthesized as orotate, attached to ribose phosphate, and then converted to the common pyrimidine nucleotides required in nucleic acid synthesis. Although the free bases are not intermediates in the de novo pathways, they are intermediates in some of the salvage pathways.
Several important precursors are shared by the de novo pathways for synthesis of pyrimidines and purines. 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.
First, there is evidence, especially in the de novo purine pathway, that the enzymes are present as large, multienzyme complexes in the cell, a recurring theme in our discussion of metabolism. Second, the cellular pools of nucleotides (other than ATP) are quite small, perhaps 1% or less of the amounts required to synthesize the cell’s DNA.
Therefore, cells must continue to synthesize nucleotides during nucleic acid synthesis, and in some cases, nucleotide synthesis may limit the rates of DNA replication and transcription. Because of the importance of these processes in dividing cells, agents that inhibit nucleotide synthesis have become particularly important in medicine.
- Nucleotides have a variety of important functions in all cells. They are the precursors of DNA and RNA. They are essential carriers of chemical energy—a role primarily of ATP and to some extent GTP. They are components of the cofactors NAD, FAD, Sadenosylmethionine, and coenzyme A, as well as of activated biosynthetic intermediates such as UDP-glucose and CDP-diacylglycerol. Some, such as cAMP and cGMP, are also cellular second messengers.
DE NOVO BIOSYNTHESIS
The detailed pathway of purine biosynthesis was worked out primarily by Buchanan and G. Robert Greenberg in the 1950s.
- In the first committed step of the pathway, an amino group donated by glutamine is attached at C-1 of PRPP. The resulting 5- phosphoribosylamine is highly unstable, with a half-life of 30 seconds at pH 7.5. The purine ring is subsequently built up on this structure.
- The second step is the addition of three atoms from glycine. An ATP is consumed to activate the glycine carboxyl group (in the form of an acyl phosphate) for this condensation reaction. The added glycine amino group is then formylated by N 10 -formyltetrahydrofolate, and a nitrogen is contributed by glutamine.
- Before dehydration and ring closure yield the five-membered imidazole ring of the purine nucleus, as 5- aminoimidazole ribonucleotide.
- At this point, three of the six atoms needed for the second ring in the purine structure are in place. To complete the process, a carboxyl group is first added. This carboxylation is unusual in that it does not require biotin, but instead uses the bicarbonate generally present in aqueous solutions.
- A rearrangement transfers the carboxylate from the exocyclic amino group to position 4 of the imidazole ring.
- Aspartate now donates its amino group in two steps, formation of an amide bond, followed by elimination of the carbon skeleton of aspartate (as fumarate).
- The final carbon is contributed by N 10 -formyltetrahydrofolate, and a second ring closure takes place to yield the second fused ring of the purine nucleus . The first intermediate with a complete purine ring is inosinate (IMP).
Conversion of inosinate to adenylate requires the insertion of an amino group derived from aspartate, this takes place in two reactions similar to those used to introduce N-1 of the purine ring. A crucial difference is that GTP rather than ATP is the source of the high-energy phosphate in synthesizing adenylosuccinate. Guanylate is formed by the NAD+ -requiring oxidation of inosinate at C-2, followed by addition of an amino group derived from glutamine. ATP is cleaved to AMP and PPi in the final step.
The common pyrimidine ribonucleotides are cytidine 5′-monophosphate (CMP; cytidylate) and uridine 5′-monophosphate (UMP; uridylate), which contain the pyrimidines cytosine and uracil. De novo pyrimidine nucleotide biosynthesis proceeds in a somewhat different manner from purine nucleotide synthesis; the six-membered pyrimidine ring is made first and then attached to ribose 5-phosphate. Required in this process is carbamoyl phosphate, also an intermediate in the urea cycle.
- Carbamoyl phosphate reacts with aspartate to yield N-carbamoylaspartate in the first committed step of pyrimidine biosynthesis (Fig. 22-38). This reaction is catalyzed by aspartate transcarbamoylase.
- By removal of water from N-carbamoylaspartate, a reaction catalyzed by dihydroorotase, the pyrimidine ring is closed to form L-dihydroorotate. This compound is oxidized to the pyrimidine derivative orotate, a reaction in which NAD+ is the ultimate electron acceptor.
- Once orotate is formed, the ribose 5-phosphate side chain, provided once again by PRPP, is attached to yield orotidylate . Orotidylate is then decarboxylated to uridylate, which is phosphorylated to UTP. CTP is formed from UTP by the action of cytidylate synthetase, by way of an acyl phosphate intermediate (consuming one ATP). The nitrogen donor is normally glutamine, although the cytidylate synthetases in many species can use directly.
REGULATION OF DE NOVO PATHWAY
- Three major feedback mechanisms cooperate in regulating the overall rate of de novo purine nucleotide synthesis and the relative rates of formation of the two end products, adenylate and guanylate.
- The first mechanism is exerted on the first reaction that is unique to purine synthesis: transfer of an amino group to PRPP to form 5-phosphoribosylamine. This reaction is catalyzed by the allosteric enzyme glutamine-PRPP amidotransferase, which is inhibited by the end products IMP, AMP, and GMP. AMP and GMP act synergistically in this concerted inhibition. Thus, whenever either AMP or GMP accumulates to excess, the first step in its biosynthesis from PRPP is partially inhibited.
- In the second control mechanism, exerted at a later stage, an excess of GMP in the cell inhibits formation of xanthylate from inosinate by IMP dehydrogenase, without affecting the formation of AMP. Conversely, an accumulation of adenylate inhibits formation of adenylosuccinate by adenylosuccinate synthetase, without affecting the biosynthesis of GMP. When both products are present in sufficient quantities, IMP builds up, and it inhibits an earlier step in the pathway; this is another example of the regulatory strategy called sequential feedback inhibition. In the third mechanism, GTP is required in the conversion of IMP to AMP, whereas ATP is required for conversion of IMP to GMP, a reciprocal arrangement that tends to balance the synthesis of the two ribonucleotides.
- The final control mechanism is the inhibition of PRPP synthesis by the allosteric regulation of ribose phosphate pyrophosphokinase. This enzyme is inhibited by ADP and GDP, in addition to metabolites from other pathways for which PRPP is a starting point
- Regulation of the rate of pyrimidine nucleotide synthesis in bacteria occurs in large part through aspartate transcarbamoylase (ATCase), which catalyzes the first reaction in the sequence and is inhibited by CTP, the end product of the sequence. The bacterial ATCase molecule consists of six catalytic subunits and six regulatory subunits. The catalytic subunits bind the substrate molecules, and the allosteric subunits bind the allosteric inhibitor, CTP.
- The entire ATCase molecule, as well as its subunits, exists in two conformations, active and inactive. When CTP is not bound to the regulatory subunits, the enzyme is maximally active. As CTP accumulates and binds to the regulatory subunits, they undergo a change in conformation. This change is transmitted to the catalytic subunits, which then also shift to an inactive conformation. ATP prevents the changes induced by CTP
- The purine ring system is built up step by step, beginning with 5- phosphoribosylamine. The amino acids glutamine, glycine, and aspartate furnish all the nitrogen atoms of purines. Two ring-closure steps form the purine nucleus.
- Pyrimidines are synthesized from carbamoyl phosphate and aspartate, and ribose 5-phosphate is then attached to yield the pyrimidine ribonucleotides.
- Nucleoside monophosphates are converted to their triphosphates by enzymatic phosphorylation reactions. Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductase, an enzyme with novel mechanistic and regulatory characteristics. The thymine nucleotides are derived from dCDP and dUMP.
- Uric acid and urea are the end products of purine and pyrimidine degradation.
- Free purines can be salvaged and rebuilt into nucleotides. Genetic deficiencies in certain salvage enzymes cause serious disorders such as Lesch-Nyhan syndrome and ADA deficiency.
- Accumulation of uric acid crystals in the joints, possibly caused by another genetic deficiency, results in gout.
- Enzymes of the nucleotide biosynthetic pathways are targets for an array of chemotherapeutic agents used to treat cancer and other diseases.