NUCLEOTIDE METABOLISM

In humans, dietary nucleotide bases are rarely incorporated into nucleotides. As a result, humans must synthesize their own nucleotide bases (With the exception of a few parasitic prokaryotes, all organisms can synthesize nucleotides) and, although most tissues can produce at least small amounts, this tightly regulated synthesis occurs mainly in the liver.

Purine biosynthesis

Purine production occurs primarily in the liver, Purine biosynthesis begins with ribose-5-phosphate (R5P, a product of the pentose phosphate pathway), and ends with the hypoxanthine containing inosine monophosphate. Both of the first two reactions in the purine biosynthetic pathway are tightly regulated.
The first reaction of the purine synthesis pathway, catalyzed by ribose 5-phosphate phosphoribosyl synthetase (PRPS), produces phosphoribosylpyrophosphate (PRPP). PRPP is not strictly committed to purine biosynthesis but also used for other processes (such as pyrimidine biosynthesis, histidine biosynthesis, and nucleotide base salvage) but its availability is critical for purine biosynthesis.

The second step, the loss of the PRPP pyrophosphate, and the addition of an amino group from glutamine, is catalyzed by PRPP amidotransferase (PPAT). This step is a specific step for purine biosynthesis.

The pathway includes nine additional enzymatic reactions. It uses R5P as a platform for the construction of the purine ring structure. 5-membered ring are firstly synthetized then the six-membered ones.

The product of the purine biosynthesis pathway is inosine monophosphate (IMP), which contains the base hypoxanthine. IMP does not accumulate and is rapidly converted to adenosine monophosphate (AMP) and guanosine monophosphate (GMP). This synthesis is tightly regulated: the first step of each pathway is inhibited by the final product (IMP dehydrogenase by GMP, and adenylosuccinate synthase (ADSS) by AMP), and both ATP and GTP are required (for GMP synthesis and AMP synthesis respectively).
Fumarate is generated during the conversion of IMP to AMP and can be incorporated in the TCA cycle.

The purine nucleoside monophosphates need to be converted to nucleoside triphosphates. Two specific enzymes for nucleoside monophosphate catalyze the monophosphate conversion to diphosphate (adenylate kinase is specific for AMP and guanylate kinase is specific for GMP). A  nucleoside diphosphate kinase allows terminal phosphate donation from any NTP to any NDP. Because free ATP is present in higher levels than any other nucleotide in cells, ATP is usually used as the phosphate donor but GTP can be occasionally used.

Pyrimidine biosynthesis

In contrast to purines that are synthesized by building the ring system on the ribose, the pyrimidine ring is constructed first, then followed by attachment of the pyrimidine base to ribose. In both purine and pyrimidine synthesis, PRPP is used as the ribose donor, but that occurs at a different stage of the pathway.

The first step of the pyrimidine synthesis pathway consists in bicarbonate condensation by cytosolic carbamoyl phosphate synthetase II (CAD II) with a nitrogen derived from glutamine to form carbamoyl phosphate (animals display two separate pools of carbamoyl phosphate: one mitochondrial used for the urea cycle, the second cytosolic for pyrimidine synthesis). To form the pyrimidine ring skeleton, an aspartate is added by CAD II in a second step.

The ribose ring is not added until pyrimidine orotic acid synthesis is complete. This orotic acid is then attached to PRPP with release of pyrophosphate. Uridine monophosphate (UMP) is then the first “completed” product.

UMP can then be phosphorylated to produce UDP. UDP will act as a branch point: it can be converted to UTP and used as a nucleotide, or it can serve as a substrate for the synthesis of the two other major pyrimidine nucleotides, CTP and TTP.

CTP is produced from UTP by using glutamine as a nitrogen donor. In contrast, thymidine monophosphate (TMP) is produced from UDP. Since thymidine is only present in DNA, the first step in the conversion of UDP to TMP is catalyzed by ribonucleotide reductase, which removes the 2´-hydroxyl, to create the deoxynucleotide. The dUDP (and any dUTP) formed is rapidly converted to dUMP to prevent the inadvertent incorporation of deoxyuridine into DNA. Once formed, dUMP is methylated by thymidilate synthase (TYMS) to produce TMP. This enzyme uses N5,N10-methylene tetrahydrofolate (a derivative of folic acid, B9 vitamin) to release dihydrofolate that then must be reduced in THF  by dihydrofolate reductase (DHFR), a crucial step for DNA synthesis.

Salvage pathways

As humans do not use dietary nucleotides for nucleic acid synthesis, they do recycle most nucleotides and free nucleotide bases in circulation through the “purine salvage pathway”.

Free purines are attached to the ribose ring (using PRPP as the base acceptor; the salvage pathway decreases the levels of PRPP, and therefore decreases the rate of purine synthesis) by two enzymes: adenine is attached by adenine phosphoribosyl transferase (APRT) and both hypoxanthine and guanine are attached by hypoxanthine-guanine phosphoribosyl transferase (HPRT).

A second pathway allows recycling of nucleosides by initiating the process of adding the 5´-phosphates. Adenosine kinase (ADK) is specific for adenosine and produced AMP, an equivalent reaction occuring for guanosine.

In contrast to free purines, salvage of free pyrimidines is a very minor pathway in most animals. The nucleoside derivatives of the pyrimidines are readily converted to nucleotides and used by cells. Thymidine kinase (TDK) is also important for DNA synthesis in many cells for this reason.

Degradation pathways

The pyrimidine ring can be completely degraded. Uracil and cytosine are converted to beta-alanine, which is required for coenzyme A synthesis. The beta-alanine can also be converted to malonyl-CoA, and used for fatty acid synthesis. Thymine is converted to beta-amino isobutyrate, which can be converted to methylmalonyl-CoA, and then to succinyl-CoA.

In contrast to the human ability to perform pyrimidine degradation, humans cannot break down the purine ring. The primary pathway for adenine nucleotide breakdown is via adenosine (and deoxyadenosine). These compounds must be converted to inosine by adenosine deaminase (ADA). Alternatively, AMP can be converted to IMP by AMP deaminase (AMPD).

Inosine and IMP production display two roles:

– it allows the conversion of adenosine to guanosine if necessary,

– it allows the generation of fumarate that will enter the TCA cycle. Under low energy conditions, muscle depends on the AMP/IMP cycle as a method for generation of some TCA cycle intermediates. An increase in muscle AMP levels is frequently a signal that energy generation by the TCA cycle is insufficient. The muscle can then use the AMP/IMP cycle to generate additional TCA cycle intermediates to increase its TCA cycle capacity

At the end, inosine can be transformed to hypoxanthine then xanthine through combined action of purine nucleoside phosphorylase (PNP) and xanthine oxidase (XDH), the latter being able to transform xanthine into uric acid, the final product of purine degradation pathway.