Nucleotide Metabolism Purines The basic structure of a purine is two-ring system that has three carbons (2,6,8) which can be oxidized. Hypoxanthine (one (6) carbon oxidized), xanthine (two (2,6) carbons oxidized), and uric acid (three (2,6,8) carbons oxidized) are all purines. Purines are synthesized bound to ribose-5-phosphate. R-5-P comes from glucose via the HMP shunt pathway. The first step in purine synthesis is the addition of two additional phosphate groups (from ATP) to R-5-P, forming PRPP (via PRPP synthetase). Purine Ring Synthesis: The purine ring is synthesized de novo in a piece by piece mechanism through nine steps. The first step is the control point involves a transfer of an NH2 group from glutamine converting it to glutamate (amidotransferase), forming PR-1 amine. Nitrogens in the final compound, which is IMP (inosine monophosphate), are derived from glutamine (2), glycine, and aspartate. The carbons of IMP are derived from glycine (2), CO2, and THF (2). IMP is a hypoxanthine nucleotide. Purine nucleotide biosynthesis occurs mainly in the liver. AMP and GMP are synthesized from IMP. AMP inhibits adenylosuccinate synthetase; GMP inhibits IMP dehydrogenase, controlling the pathway. Both AMP and GMP synergistically inhibit the synthesis of IMP at the amidotransferase step. To synthesize AMP from IMP, an amino group from aspartate is added (via adenylosuccinate synthetase). The resulting adenylosuccinate is cleaved by adenylosuccinase releasing fumarate and AMP. To synthesize GMP from IMP, IMP is first oxidized to XMP using IMP dehydrogenase, and then an amino group is transferred from glutamine to XMP (using an amidotransferase) resulting in GMP. The nucleotide mono phosphates (NMPs) can be phosphorylated to yield NDPs. These nucleotide 5-monophosphate kinases that carry out these reactions are specific for the base only (for example, the GMP kinase will phosphorylate both GMP and dGMP), and get the phosphate from ATP. These NDPs can be further phosphorylated by nucleotide 5-diphosphate kinase, which will work for all bases and sugars. However, ATP is made using the oxidative phosphoryalation method, and not these kinases. Purines are not only made by the body, they are also catabolized. This involves removal of the NH2 group (via a deaminase), removal of the phosphate group (via a phosphodiesterase), and removal of the sugar (via a purine nucleotide phosphorylase). The remaining purine ring is fully oxidized to the uric acid form, and is excreted. **SCID, severe combined immunodeficiency disorder, is genetic defect in adenosine deaminase. This causes B and T cell abnormalities and ultimately immunodeficiency. **Gout is a buildup of uric acid crystals. These crystals can precipitate in the joints and cause pain, and can also be a component of kidney stones. Primary gout is caused by the overproduction of nucleotides. Secondary gout is the buildup of uric acid crystals secondary to another disease where there is higher cell (and thus nucleotide) turnover, overwhelming the system, such as cancer. **If there is a defect in liver G-6-Pase there will be an increase in G-6-P, of which only a finite amount can be stored as glycogen. Therefore, there will be an increase in nucleotide biosynthesis, and thus breakdown. Symptoms of G-6-Pase deficiency include hypoglycemia and gout. However, sometimes the body does not want to break down purine nucleotides all the way to uric acid. Fortunately, purine salvage pathways exist. PRPP can be added to hypoxanthine and guanine to form IMP and GMP, respectively, via hypoxanthine- guanine phosphoribosyl transferase (HGPRT). PRPP can be combined with adenosine to form AMP using adenosine phosphoribosyl transferase (APRT). **Lesch-Nyhan disease is a genetic defect in HGPRT. Symptoms include self- mutilation, hostile tendencies, and chewing fingers and lips. HGPRT is involved in an important salvage pathway in the brain. Pyrimidine In contrast to the purine ring, which is synthesized on R-5-P (actually PRPP), pyrimidine ring biosynthesis occurs and R-5-P is added later. The pyrimidine ring begins as carbamoyl phosphate and aspartate. Carbamoyl phosphate for pyrimidine synthesis is made by CP synthase II, converting CO2 and the NH3 from glutamine into carbamoyl phosphate, at the expense of ATP. This carbamoyl phosphate can combine with aspartate in the first rate limiting step, catalyzed by transcarbamoylase. The end product of this synthesis is UMP. UTP and UDP are negative modulators of transcarbamoylase, and ATP is a positive modulator, thus tying the purine and pyrimidine biosynthesis pathways together. UMP is converted to UTP (using UMP kinase and NDP kinase). UTP is then converted to CTP by CTP synthetase (taking an NH2 from glutamine and a phosphate from ATP). We have previously covered how the body makes NTPs and NDPs. For DNA synthesis, the cell needs to convert these NDPs to dNDPs. This is done via rib nucleotide reductase, whose substrates include UDP, CDP, ADP, and GDP. A small dithiol protein, thioredoxin, serves as the reducing agent. Oxidized thioredoxin is regenerated by oxidizing NADPH to NADP. The nucleotide TTP (thymidylate) is synthesized from dUMP. A one carbon moiety (methylene) is added to dUMP via THF. The conversion of dUMP to dTMP is catalyzed by thymidylate synthase and produces FH2 from methylene FH4.
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