Chapter 27 - Columbia University
Synthesis of purine ribonucleotides
IMP is synthesized from ribose 5-phosphate. There are 11 reactions in the formation of IMP. IMP is converted to GMP and AMP with the help of ATP and GTP respectively. Nucleoside monophosphates are converted to nucleoside diphosphates by base specific monophosphate kinases. Purine nucleotide synthesis is regulated by feedback inhibitor – AMP, GMP and IMP. An important regulatory factor is the availability of PRPP. Salvage pathway for purines is observed in RBC and the brain. Free purines are salvaged by APRTase and HGPRTase enzymes
Synthesis of pyrimidine ribonucleotides
Pyrimidine ring is synthesized as free pyrimidine and then it is incorporated into the nucleotide. 6 reactions are involved in the synthesis of UMP. UDP and UTP are synthesized from UMP with the help of ATP. CTP is formed by adding an amino group from glutamine. Pyrimidine can also be salvaged using PRPP. In orotic aciduria, excretion of large amount of orotic acid is observed. It results from the deficiency of either orotate phospho ribosyl transferase or OMP decarboxylase.
The Synthesis and Degradation of Nucleotides
We wanted to investigate why the CTP pool is so low in the bloodstream form of T. brucei. This low level of CTP could be the consequence of deficient synthesis or of excessive degradation of CTP. First, we determined the rate of de novo CTP synthesis. Trypanosomes that were offered tritiated uracil in the culture medium incorporated label into both the UTP and the CTP pools (Fig. A). Thus we can conclude that trypanosomes have a de novo pathway for CTP synthesis. The accumulation of label in the UTP pool was rapid and was almost saturated after 5 min, whereas the CTP pool was labeled at a much slower rate. This observation suggested that slow de novo synthesis could be responsible for the low CTP level.
The synthesis of the pyrimidines CTP and UTP occurs in the cytoplasm and starts from the condensation of aspartate with carbamoyl-phosphate to form orotic acid.
KEGG PATHWAY: Metabolic pathways - Reference pathway
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, MIT, Cambridge, MA
The synthesis of brain phosphatidyl choline may utilize three circulating precursors: choline; a pyrimidine (e.g., uridine, converted via UTP to brain CTP); and a PUFA (e.g., docosahexaenoic acid); phosphatidylethanolamine may utilize two of these, a pyrimidine and a PUFA.
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We also wanted to know whether the size of the CTP pool was controlled by a high degradation rate. We therefore checked the label in the first two degradation products of CTP catabolism, cytidine and uridine, by HPLC chromatography. No radioactivity was detectable in these compounds. On the contrary, label was readily incorporated into nucleic acids (Fig. B). Considering the detection limit of our experiments, we can conclude that at least 95% of the CTP and UTP in trypanosomes was used for nucleic acid synthesis, whereas less than 5% was degraded. A rapid degradation is therefore not responsible for the low CTP pool in T. brucei.
Statistical Techniques | Statistical Mechanics
The absolute dependence of T. brucei on de novo synthesis for CTP production might be exploited for chemotherapy purposes, because the host can be rescued by cytidine. We tested two glutamine analog CTP synthetase inhibitors, DON and acivicin, and we measured how the NTP pools of cultured bloodstream forms of T. brucei changed during the 1-h incubation period. DON specifically lowered the CTP pool, and acivicin also lowered the GTP pool (Fig. A). None of the drugs affected the ATP or the UTP pools (data not shown). The inclusion of 160 μM guanine in the growth medium abolished the effect by acivicin on the GTP pool. Because the CTP level was still low, we conclude that the inhibition of CTP synthesis by acivicin was direct and was not due to an allosteric effect by GTP. The result with acivicin is in agreement with the situation in mammalian cells, where it is established that the main targets are CTP synthetase and GMP synthase (). Trypanosomes lack the enzymes in the de novo purine biosynthesis pathway up to IMP but contain later enzymes such as GMP synthase (). It is therefore not surprising that GTP synthesis was affected by acivicin. The inclusion of 5 mM cytidine or 1 mM cytosine in the growth medium did not affect the inhibition of CTP synthesis by DON or acivicin (data not shown).