Metabolism of Purine and Pyrimidine Prof.Orhan CANBOLAT ; MD , PhD

There are two major purines, adenine (A) and guanine (G), and three major pyrimidines, cytosine (C), uracil (U), and thymine (T).

Purine and Pyrimidine A nucleoside is formed from the linkage of a sugar with a nitrogen-containing base., 1. The bases that make up the physiologically relevant nucleosides all have ring structures. a. The purines adenine, guanine, and inosine have a double- ring system. b. The pyrimidines cytosine, thymine, and uracil have six- membered ring structures. 2. Ribose and 2-deoxyribose are the main sugars found in nucleosides and nucleotides. Nucleotides ; one, two, or three phosphate groups have been added to the sugar.

Nucleosides - Nucleotides

Biomedical Importance DNA – RNA ( Nucleic acid metabolism ) ATP – GTP (Energy Metabolism ) NADH – FADH( Co- enzymes ) S- Adenosyl – methionine (donor for methyl group ) UDP – glucronic acids ( donor for conjugation ; bilirubin glucronid ) cAMP – cGMP ( Hormone Signal ) Anticancer Drugs ( Mtx , 5Flu, 6-TG , 6-MG ) Antiviral drugs (Aciclovir , Lamivudine  ) Gout Disease Treatment ( Allopurinol ) Human diseas that involve purine and pyrimidine (Gout Disease , Lesh Nyhan Synrome , ADA Deficiency , Orotic aciduria )

Purines & Pyrimidines Are Dietarily Nonessential Human tissues can synthesize purines and pyrimidines from amphibolic intermediates. Ingested nucleic acids and nucleotides, which therefore are dietarily nonessential, are degraded in the intestinal tract to mononucleotides, which may be absorbed or converted to purine and pyrimidine bases.

The sources of nitrogen and carbon atoms of purine ring 4 N C 1 2 3 5 6 8 9 7 GLİSİN N5, N10 methenilH4folat N10-Formiltetrahidrofolat GLUTAMAT CO2 ASPARTAT The sources of nitrogen and carbon atoms of purine ring

PRPP Biosynthesis of Purine Nucleotides ; De- Novo Mg ATP AMP Ribose 5-phosphate derived from the pentose phosphate pathway or from dietary sources is the starting material that eventually gives rise to inosine monophosphate (IMP) Biosynthesis of Purine Nucleotides ; De- Novo Mg ATP AMP PRPP Synthetase PRPP Ribose 5-Phosphate key regulatory enzyme The first step creates the multi-purpose intermediate 5-phosphoribosyl-1-pyrophosphate (PRPP).

In prokaryotes, is catalyzed by a different polypeptide by contrast, in eukaryotes, the enzymes are polypeptides with multiple catalytic activities

FOLIC ACID DEFICIENCY • Decreased levels of folate coenzymes needed for various reactions of de novo purine synthesis and thymine synthesis produce shortages of deoxyribonucleotides and consequent impaired DNA synthesis in many tissues. • Blood levels of folic acid may become inadequate due to dietary insufficiency or poor absorption due to intestinal problems or alcoholism. • Folate coenzyme concentrations may also decline as a result of treatment with drugs that inhibit dihydrofolate reductase, eg, methotrexate . • Patients with folic acid deficiency may have diarrhea and nausea, but the principal symptoms are weakness and easy fatigability due to megaloblastic anemia arising from impaired cell division in the bone marrow. • Folate deficiency during pregnancy is a major contributor to neural tube defects because of the critical role of folate in neuronal development. • Folate supplementation of food in the United States is expected to reduce folate-associated birth defects by up to 70%.

Biosynthesis of Purine Nucleotides ; Salvage Salvage Reactions“ Convert Purines & Their Nucleosides to Mononucleotides

Salvage of purine nucleosides Free purines may be joined with PRPP to produce mononucleotides by one of two enzymes. The reaction shown below is catalyzed by adenine phosphoribosyltransferase (APRT). Adenine + PRPP → AMP + Ppi 2. The following reactions are catalyzed by , hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Hypoxanthine + PRPP → IMP + PPi Guanine + PRPP → GMP + Ppi Salvage of purine nucleosides is achieved by phosphorylation with ATP as the phosphate donor.

LESCH-NYHAN SYNDROME • Lesch-Nyhan syndrome is an X-linked disorder arising from deficiency of HGPRT, which results in failure to salvage hypoxanthine and guanine to the corresponding nucleotides IMP and GMP. • Inability to utilize PRPP in the salvage pathway leads to PRPP accumulation, which, in conjunction with low levels of IMP and GMP, causes chronic allosteric activation of PRPP glutamyl amidotransferase and excessive purine synthesis. • The excess purines are degraded to uric acid causing increased blood levels of this metabolite (hyperuricemia) and deposition of sodium urate crystals in the joints and kidneys. • Patients with Lesch-Nyhan syndrome experience gout-like episodes of joint pain and kidney stones as well as severe neurologic problems, including self-mutilation, spastic movements, and mental retardation. •

Degradation of purine nucleotides to uric acid

GMP Ksantin Oksidaz GUANOZİN ADP GUANİN R-1P PNP H2O2 . O2 5’NT Pi ADA NH3

Feed back Regulation AMP and GMP also inhibit AMP & GMP Feedback-Regulate Their Formation from IMP GMP , ADP feedback-inhibits PRPP glutamyl amidotransferase ADP ,GDP feedback-inhibits PRPP synthetase AMP and GMP also inhibit hypoxanthine-guanine phosphoribosyltransferase,

? Hyperuricemia

inflammation (gouty arthritis) are the hallmarks of gout. •Hyperuricemia and chronic or episodic joint pain due to deposition of sodium urate crystals and consequent inflammation (gouty arthritis) are the hallmarks of gout. • Uric acid is minimally water-soluble and most cases of gout arise from inadequate excretion by the kidneys ,leading to build-up of uric acid and precipitation of urate stones in the kidneys and extremities. • The joints of the hands and feet are prone to accumulation of crystals because of reduced solubility of sodium urate at the slightly cooler temperature of the extremities.

1.PRPP synthetase ATP AMP Riboz 5-P PRPP PRPP sentetaz ÜRİK ASİT

Ischemia - Reperfusion ATP YIKIMI ARTIŞI ADP HİPOKSANTİN H2O2 KO O2 . KO ÜRİK ASİT

3. Von Gierke’s Disease G-6-Paz G-6-P GLUKOZ HMŞ glukoneogenez HMŞ R-5-P üretiminde artış ÜRİK ASİT ARTIŞI

Immun system deficiency 5’AMP AMP dATP 5’NT ADENOZİN ADENOZİN RNdiP ADA Ribo- nükleotid redüktaz İNOZİN İNOZİN dRNdiP PNP DNA sentezinin bozulması HİPOKSANTİN T ve B hücre Immun system deficiency

HİPOKSANTİN KSANTİN ÜRİK ASİT KSANTİNÜRİ Ve KSANTİN TAŞI HİPOÜRİSEMİ KSANTİN OKSİDAZ KSANTİN KSANTİNÜRİ Ve KSANTİN TAŞI KSANTİN OKSİDAZ ÜRİK ASİT HİPOÜRİSEMİ

Ribonucleotide Reductase

Biosynthesis of Pyrimidine Nucleotides 6 N C 3 2 1 5 4 ASPARTAT GLUTAMİN HCO3

Biosynthesis of pyrimidine nucleotides The pyrimidine ring is synthesized first and is then attached to ribose 5-phosphate to eventually produce the nucleotide uridine 5′-monophosphate (UMP). Biosynthesis of pyrimidine nucleotides

Carbamoyl phosphate synthetase II ; CPS-II An ammonium ion contributed by glutamine is combined with bicarbonate (derived from dissolved CO2) in a two-step reaction that requires hydrolysis of two molecules of ATP. b. This complex reaction is catalyzed by carbamoyl phosphate synthetase II(CPS-II). c. CPS-II, the critical enzyme regulating the pyrimidine synthetic pathway, is activated by ATP and PRPP and feedback-inhibited by the end product UTP. CPS-II, a cytosolic enzyme, is different from the mitochondrial enzyme of the urea cycle CPS-I.

Synthesis of dTTP

Mechanism of Tymidylate synthesis Deoxythymidylate (dTMP) is formed from 2′-deoxyuridylate (dUMP) in a one carbon transfer by thymidylate synthetase 1. The donor coenzyme for the one-carbon transfer is N5,N10-methylene tetrahydrofolate (N5,N10-methylene THF); simultaneous reduction to a methyl group leaves dihydrofolate (DHF) as byproduct. 2. N5, N10-methylene THF is regenerated from DHF by a series of reactions, one of which involves dihydrofolate reductase.

INHIBITORS OF dTMP SYNTHESIS AS ANTICANCER AGENTS • Several drugs that interfere with production of dTMP by blocking the reaction catalyzed by thymidylate synthetase are inhibitors of DNA synthesis and cell proliferation. The thymine analog 5-fluorouracil (5-FU) is converted to 5-fluoro-dUMP, which acts as a suicide inhibitor of thymidylate synthetase. • Methotrexate is a folate analog that acts as a potent competitive inhibitor of dihydrofolate reductase,causing a decreased supply of THF coenzymes needed by thymidylate synthetase.

Control of Pyrimidine Nucleotide İnhibition of CPSII by UMP and UTP Activation of PRPP and ATP CO2 + Glutamin + ATP UMP ,UTP Karbomoil fosfat Aspartat CTP N-karbomoil aspartat UMP UTP CTP

Catabolism of pyrimidine

Orotic aciduria Mutation of one of the two enzyme activities of UMP synthase leads to orotic aciduria, characterized by accumulation of its first substrate orotic acid and insufficient levels of the product UMP, which reduces availability of uridine triphosphate (UTP) and cytidine triphosphate (CTP) for use in nucleic acid synthesis. • Patients with orotic aciduria excrete large amounts of orotic acid in their urine, and they exhibit lethargy, weakness, severe anemia, and growth retardation. • This autosomal recessive disorder can be treated by feeding a diet rich in uridine, which is salvaged to UMP and finally to UTP.

İnhereted disorders of pyrimidine metabolism Clinical Disorder Defective enzmyme Orotic aciduria Type I Orotate phosphoribosyl transferase Orotic acid crystallüria , megaloblastic anemia ,immu deficiency Orotic aciduria Tip II orotidate decarboxilase Ornitihine transcarbamoylase deficiency Ornitihine transcarbamoylase Mild orotic aciduria