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primidine metabolism.pptx
- 1. http://www.free-powerpoint-templates-design.com PYRIMIDINE METABOLISM Dr Manoj Acharya Department of Biochemistry
- 2. Overview • Structural aspects and history • de novo synthesis of Pyrimidine bases. • Regulation of synthesis. • Synthesis of deoxy pyrimidine bases. • Catabolism of pyrimidine bases.. • Disorder of pyrimidine metabolism.
- 3. Introduction
- 4. Structure of Pyrimidine bases • Pyrimidine bases: CUT • Pyrimidine nucleotides are - CMP - UMP - TMP
- 5. History Adolf Pinner first proposed the name “pyrimidine” in 1885. Discovered by Albrecht Kossel and Albert Neumann in 1894, it was hydrolyzed from calf thymus. unstable and can change to uracil (spontaneous deamination). Demethylated form of thymine. Discovered in 1900 by Alberto Ascoli. Planar, unsaturated compound, absorbs light. Only in RNA. First isolated in 1893 by Albrecht Kossel and Albert Neumann from calf thymus glands, hence its name. May be derived by methylation of uracil at the 5th carbon. Only in DNA.
- 6. Nomenclature of bases, nucleosides and nucleotides
- 7. Synthesis: de Novo or Salvage • de Novo Synthesis: nucleotide bases are assembled from simpler compounds. • Salvage Pathway : preformed bases are recovered and reconnected to ribose unit.
- 8. • Denovo pathways : synthesis from aspartate, glutamine, ribose 5-phosphate, CO2 and ATP. • Different from purine de novo synthesis, first pyrimidine ring is assembled and linked to ribose phosphate. • Ammonia used here is usually produced from hydrolysis of sidechain of glutamine.
- 9. 1. Carbamoyl phosphate synthetase • First Step: Activation • Bicarbonate and ammonia with 2 ATP gives carbomyl phosphate in multistep, catalyzed by carbomyl phosphate synthetase II (CPSase II).
- 10. Structure of CPSase II • 2 chain with 3 catalytic domains: • Compartmentation provides 2 independent pools of carbamoyl phosphate.
- 11. 2. Aspartate transcarbamoylase • Addition of aspartate. • Committed step in pyrimidine biosynthesis. • Inhibited by CTP (end product), • Activated by ATP in bacteria.
- 12. 3. Dihydro-orotase: Cyclization • Formation of dihydro-oratate • Closure of ring with loss of water. • First 3 enzymes are multifunctional polypeptides (CAD). • Ensures efficient channeling for high energy carbamoyl phosphate to pyrimidine biosynthesis.
- 13. 4. Dihydroorotate dehydrogenase • Dehydrogenase cause formation of double bond. • Occurs in inner mitochondrial membrane • The ultimate electron acceptor is NAD+
- 14. 5,6. UMP Synthase • Orotate phosphoribosyl transferase and OMP decarboxylase (bifunctional enzyme) are separate domains of a single polypeptide—UMP synthase. • Addition of ribose 5-phosphate from PRPP to form orotidylate. • Removal of carboxylic group from orotidylate form uridylate (UMP)
- 16. 7. Formation of CTP and TMP THF Serine hydroxylmeth yl transferase Glycine Serine Dihydrofolat e reductase Methotrexate Trimethoprim 5-Fluoro uracil Hydroxy urea
- 17. Salvage of Pyrimidines • Few Pyrimidine bases are salvaged in human cells. • Salvaged by Nucleoside kinases. • Salvage pathway is used for treatment of Hereditary orotic aciduria.
- 18. Regulation of Synthesis - CPSase II: inhibited by UTP and purine nucleotides, activated by PRPP. - Aspartate transcarbamoylase is inhibited by CTP but activated by ATP in bacteria
- 19. Ribonucleotide Reductase • Key enzyme for evolution mediates the synthesis of deoxyribonucleotides. • Substrates for DNA synthesis RNA DNA
- 20. Coordinate regulation of Purine and pyrimidine synthesis • For each mol of deoxy purine nucleotide, deoxy pyrimidine nucleotide needs to be synthesized, which is regulated by ribonucleotide reductase allosteric regulation mechanism.
- 21. Biosynthesis of Thymidylate • DNA contains thymine rather than uracil and the de novo pathway to thymine involves only deoxyribonu- cleotides. • Immediate precursor of thymidylate (dTMP) is dUMP. • Pathway to dUMP begins with formation of dUTP, either by deamination of dCTP or by phosphorylation of dUDP.
- 22. Conversion of dUMP to dTMP is catalyzed by thymidylate synthase. - A one-carbon unit at the hydroxymethyl oxidation level is transferred from N5, N10- Methylenetetrahydrofolate to dUMP, then reduced to a methyl group. - Reduction occurs at the expense of oxidation of tetrahydrofolate to dihydrofolate, which is unusual in tetrahydrofolate- requiring reactions. - Dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase
- 23. Mono, di and triphosphates are interconvertible • Active forms are NDP and NTPs. • Monophosphate to diphosphate by nucleoside monophosphate kinases with ATP as phosphoryl donor (only base specific). UMP + ATP UMP Kinase UDP + ADP • Diphosphate and Triphosphate are interconverted by nucleoside diphosphate kinase (nonspecific). XDP + YTP nucleoside diphosphate kinase XTP + YDP
- 24. Chemotherapeutic Agents Target Enzymes • Useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, enzymes that provide only cellular pathway for thymine synthesis. • Fluorouracil is an inhibitor that acts on thymidylate synthase, chemotherapeutic agent. • Fluorouracil itself is not the enzyme inhibitor. • In the cell, salvage pathways converts it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme
- 25. • Methotrexate is an inhibitor of dihydrofolate reductase. • This folate analog acts as a competitive inhibitor; the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. • Aminopterin is a related compound that acts similarly. • Trimethoprim, an antibiotic binds to bacterial dihydrofolate reductase nearly 100,000 times better than to the mammalian enzyme.
- 26. Catabolism of Pyrimidine • Unlike purine, not cleaved, pyrimidine ring is opened and degraded to soluble products like β-alanine, β-aminisobutyrate, NH3 and CO2.
- 27. • β-alanine excreted or converted to carnosine or anserine. • β-aminisobutyrate is excreted in urine or transaminate to from methylmalonyl semialdehyde and finally to succinyl-CoA. • Excretion of β aminoisobutyrate increase in leukemia. Fig :
- 28. Disorders of Pyrimindine metabolism • Since the product of pyrimidine catabolism are highly water soluble, overproduction of pyrimidine catabolites is rarely associated with clinically significant abnormalities. • Disorder related to Pyrimidine Synthesis - Orotic aciduria
- 29. Disorders of Pyrimindine metabolism Orotic Aciduria: Hereditary autosomal recessive disorder. • Cause: - Deficiency in UMP synthase (Orotate Phosphoribosyl Transferase and Orotidylate Decarboxylase). - Secondary to blockage of Urea cycle by Ornithine transcarbamylase deficiency distinguished by decrease in urea with hyperammonemia. • .
- 30. Two Types Type I – There is deficiency of both enzymes orotate phosphoribosyltransferrase and OMP decarboxylase Type II – Only OMP decarboxylase is deficient https://www.amjmed.com/article/0002-9343(73)90260-X/fulltext
- 31. Clinical features and treatment • There is retarded growth and megaloblastic anemia • Crystals excreted in urine • Crystalluria may cause UTI Treatment: Oral administration of uridine bypass metabolic block and provide source of pyrimidines
- 32. Reye’s Syndrome or Secondary Orotic Aciduria • This presents similar to hereditary orotic aciduria and is not due to defective pyrimidine nucleotide synthesis but defective mitochondria. • Ornithine transcarbomylase of urea cycle is defective so carbomyl phosphate diffuses out into cytosol to lead mild orotic aciduria • It is associated with high blood ammonia and glutamine levels, • In this condition ammonia metabolism is major concern. Orotic aciduria is of secondary importance.
- 34. Lab Assessment • Orotic acid can be converted to barbituric acid by bromination and reduction in presence of ascorbic acid. • Barbituric acid is coupled with p-dimethylaminobenzaldehyde to form orange product. • 5-( p-dimethylaminobenzylidene) barbituric acid, which absorbs light at 480 nm. • Orotic acid are measured by this method unless the metabolites are separated chromatographically before analysis Salerno C, Crifò C. Diagnostic value of urinary orotic acid levels: applicable separation methods. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Dec 5;781(1-2):57-71. doi: 10.1016/s1570-0232(02)00533-0. PMID: 12450653.
- 35. Drugs may precipitate Orotic aciduria • Allopurinol: alternative substrate for orotate phosphoribosyltransferase competes with orotic acid, resulting nucleotide product inhibit orotidylate decarboxylase resulting in orotic aciduria. • 6-Azauridine: by conversion to 6-azauridylate also competitively inhibits orotidylate decarboxylase, resulting orotic aciduria.
- 36. References • Lehninger Principles of Biochemistry • Harper’s Illustrated Biochemistry • Lippincott’s illustrated biochemistry • Biochemistry Stryer • https://www.amjmed.com/article/0002-9343(73)90260- X/fulltext • https://sohebbasharat.wordpress.com/2-16/02/20/orotic- aciduria • Salerno C, Crifò C. Diagnostic value of urinary orotic acid levels: applicable separation methods. J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Dec 5;781(1-2):57-71. doi: 10.1016/s1570- 0232(02)00533-0. PMID: 12450653.
- 37. Thank you
Notes de l'éditeur
- Nucleoside: Nitrogenous base + Pentose sugar Nucleotide: Nucleoside + Phosphate
- Cytidine monophosphate Uridine monophosphate Thimidine monophosphate -The specific role of the carbamoyl phosphate synthetase I enzyme is to control the first step of the urea cycle, a reaction in which excess nitrogen compounds are incorporated into the cycle to be processed.
- Deamination is the removal of an amino group from a molecule.
- The Pyrimidine ring is assembled de Novo or Recovered by salvage pathways. The framework of pyrimidine base is assembled first and then attached to ribose whereas in purine base is synthesized piece by piece directly onto ribose based structure Phosphoribosyl pyrophosphate (PRPP) ribose 5-phosphate + ATP → PRPP + AMP. Ribose sugar from HMP shunt
- deoxy pyrimidine nucleotide needs to be synthesized
- The specific role of the carbamoyl phosphate synthetase I enzyme is to control the first step of the urea cycle, a reaction in which excess nitrogen compounds are incorporated into the cycle to be processed.
- Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein. The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia.
- multi-subunit protein complex composed of 12 subunits The particular arrangement of catalytic and regulatory subunits in this enzyme affords the complex with strongly allosteric behaviour with respect to its substrates. ATCase does not follow Michaelis–Menten kinetics. Instead, it lies between its low-activity, low-affinity "tense" and its high-activity, high-affinity "relaxed" states The committed step is the one after which the substrate has only one way to go. Carbamoyl phosphate condenses with aspartate to form carbomyl aspartate.
- dihydro-oratase catalyses the ring closure by loss of water molecule The three enzymes are the domain or functional unit of the same protein.
- This reaction is catalyzed by dihydroorotate dehydrogenase Oxidized by NAD+ and getting itself reduced to the NADH
- Orotic acid is converted to Orotodine MonoPhosphate by enzyme orotate phosphoribosyl transferase and PRPP is the donor of ribose. Then OMP is decarboxylated to UMP by enzyme orotidylic acid decarboxylase. PRPP is derived from the first reaction of purine synthesis faciliated by the enzyme PRPP synthase
- Hydroxy Urea (antimetabolites): inhibition of the enzyme ribonucleotide reductase by scavenging tyrosyl free radicals. Sickle cell dz, myloproliferative dz, Psoriasis, CML etc Fluorouracil, an anticancer drug, is converted in vivo into fluorodeoxyuridylate(F-dUMP). This irreversibly inhibits thymidylate synthase after Methotrexate: Anticancer and anti rheumatic arthritis: prevent rapid turnover of immune cells. Trimethoprim- sulfamethoxazole (in bacteria): prokaryotes: UTI and mid ear infection :binds bacterial DFR 100,000X than human. The major folate present in mammalian cells is 5-methyl-tetrahydrofolate (5-MTHF), which provides methyl groups to the S-adenosylmethionine (SAM) cycle through the conversion of homocysteine (Hcy) to methionine
- Pyrimidines can be synthesized or reprocessed or salvaged from old pyrimidines. So the breakdown of DNA can lead in formation of cytosine and Thymine. Now for uracil co factor is ribose 1 phosphate. Uracil can come from RNA also. Ribose 1 phosphate and deoxyribose 1 phosphate are less amount or abundant in the cell and only less amount of pyrimidines is processed.
- For a given enzyme concentration, the value of enzyme reaction increases or decreases with increasing or decreasing substrate concentration
- Ribose sugar needs to be converted to deoxyribose sugar to make deoxy ribose phosphate for DNA synthesis which is done by the single enzyme ribonucleotide reductase. ribonucleotide reductase is the enzyme that ensures there is the balance in the synthesis of all deoxyribonucleotide triphosphate. Faciliated by formation of water molecule . Hydroxyl group of C2 carbon is converted to water facilitated by thiol group containing Thioredoxin and first of all deoxyribose diphosphate is made.
- For balance synthesis of deoxynucleotides the enzymes should be highly regulated. So positive allosteric regulator is ATP and negative modulator is dATP. When ATP gets bind to the allosteric site first of all CDP gets bind to the active site and converted as dCDP then converted to dCTP then in second dUDP gets bind to allosteric site converting to TTP. TTP now increases and then replaces dATP in the allosteric binding site then GDP will bind to the active site converting to dGDP and to dGTP then dGDP replaces dTTP in the allosteric site and ADP binds to the active site converting to dADP and dATP. Since dATP is a negative modulator of the enzyme so after it gets bind to the allosteric site the process of formation stops.
- Hydroxymethyl group- CH2OH. Dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase— a regeneration that is essential for the many processes that require tetrahydrofolate. In plants and at least one protist, thymidylate synthase and dihydrofolate reductase reside on a single bifunctional protein. The major folate present in mammalian cells is 5-methyl-tetrahydrofolate (5-MTHF), which provides methyl groups to the S-adenosylmethionine (SAM) cycle through the conversion of homocysteine (Hcy) to methionine
- It is used to treat certain urinary and middle ear bacterial infections. Aminopterin is an amino derivative of folic acid, which was once used as an antineoplastic agent in the treatment of pediatric leukemia Parasitic protists, such as the trypanosomes that cause African sleeping sickness (African trypanosomiasis), lack pathways for de novo nucleotide biosynthesis and are particularly sensitive to agents that interfere with their scavenging of nucleotides from the surrounding environment using salvage pathways. Allopurinol and several similar purine analogs have shown promise for the treatment of African trypanosomiasis and related afflictions. α-DifluoromethylOrnithine (DFMO) Inhibitor of Ornithine Decarboxylase
- Nucleotidase is the enzyme removes the phosphate group. Phosphorylase enzyme removes the sugar Deaminase (incorporation of water molecule) , Reductase
- Hydrolase (both). CO2 and NH3 comes out .. In human beings, beta-amino-iso-butyrate is trans-aminated to methylmalonate semialdehyde which is further converted to succinyl-CoA Carnosine and anserine are strong antioxidants,
- A defect in the UMP synthase results in the excretion of orotic acid in urine.
- Feeding cytidine and Uridine (Cytidine is a drug used to manage neuropsychiatric deficits associated with cerebrovascular diseases in combination with uridine) Uridine and choline work synergistically with DHA(Docosahexaenoic acid)omega 3 fatt acid to increase phosphatidylcholine formation animal muscle is naturally rich in ATP, meat (pork, beef, chicken), fish and shrimps are excellent sources of purine nucleotides; 2) Baker yeasts are naturally rich in RNA and yeast extracts are excellent sources of both purine and pyrimidine nucleotides
- Ornithine transcarbomylase
- Since histidine do not form color so if there is presence of histidine amino acid interference, bromination is required
- 6-Azauridine is a synthetic triazine analogue of uridine with antimetabolite activity