What would They Say

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What would they say If we up and ran away
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What would They Say

  1. 1. A Christmas Prayerby Robert Louis StevensonLoving God, help us to remember the birth of Jesus that we may share in the songs of the angels, the gladness of the shepherds, and the worship of the wise men.Close the door of hate and open the door of love all over the world. Let kindness come with every gift and good desires with every greeting. Deliver us from evil by the blessing which Christ brings.May our minds be filled with grateful thoughts and our hearts with forgiveness, for Jesus‟ sake.Amen.
  2. 2. Nucleotide Metabolism adenosine triphosphate Noel Martin S. Bautista, MD, MBAH Department of Biochemistry, Molecular Biology and Nutrition
  3. 3. Introduction Overview of nucleotide metabolism and biological functions of nucleotides Metabolism of purine nucleotides Clinical disorders of purine metabolism Metabolism of pyrimidine nucleotides Clinical disorders of pyrimidine metabolism Metabolism of deoxyribonucleotides Different pharmacologic agents that interfere with nucleotide metabolism 4
  4. 4. Nucleobases 5
  5. 5. base + sugar = Nucleosides have either -D-ribose or -D-deoxyribose in an N-glycosidic linkage between C-1 of the sugar and N-9 (purine) or N-1 (pyrimidine) Ribonucleosides 6
  6. 6. base + sugar = Nucleosides have either -D-ribose or -D-deoxyribose in an N-glycosidic linkage between C-1 of the sugar and N-9 (purine) or N-1 (pyrimidine) Deoxyribonucleosides 7
  7. 7. Nucleoside + PO4 = Nucleotides  have one or more phosphate groups esterified to the sugar Deoxyribonucleotides Ribonucleotides 8
  8. 8. Metabolic Functions of Nucleotides Function Selected Examples 1. Energy metabolism ATP (muscle contraction, etc) 2. Monomeric units of NTPs and dNTPs (substrates for RNA and DNA) nucleic acids Adenosine (coronary blood flow); ADP (platelet 3. Physiological aggregation); cAMP and cGMP (second mediators messengers) GTP (mRNA capping); tetrahydrobiopterin 4. Precursor function (hydroxylation of aromatic amino acids) 5. Components of NAD, FAD, FMN and coenzyme A coenzymes UDP-glucose (glycogen); CDP-choline 6. Activated (phospholipids); SAM (methylation); PAPS intermediates (sulfation) ATP (negative effector of PFK-1); AMP (positive 7. Allosteric effectors effector of phosphorylase b); dATP (negative effector of ribonucleotide reductase)overview 9
  9. 9. Nucleotide Metabolism Overview 10
  10. 10. Nucleotide Synthesis: De Novo Pathway De Novo Pathway 11
  11. 11. Nucleotide Synthesis: Salvage Pathwaycentral 12
  12. 12. PRPP: Central Metabolite 5-phospho-D-ribosyl-1-pyrophosphate (PRPP) is an intermediate in both the de novo and salvage synthesis of nucleotides synthesized from ribose-5-phosphate  from pentose phosphate pathway (HMP)  phosphorolysis of nucleosides by nucleoside phosphorylase 13
  13. 13. PRPP Synthesis  formed through the action of PRPP synthetase, which activates carbon 1 of ribose-5-phosphate by transferring to it the pyrophosphate moiety of ATP 14
  14. 14. Reactions Requiring PRPP: de novo purine nucleotide synthesis: PRPP + glutamine  5-phosphoribosylamine + glutamate + PPi salvage of purine bases: PRPP + hypoxanthine (guanine)  IMP(GMP) + PPi PRPP + adenine  AMP + PPi de novo pyrimidine nucleotide synthesis: PRPP + orotate  OMP + PPi salvage of pyrimidine bases PRPP + uracil  UMP + PPi NAD+ synthesis 15
  15. 15. Synthesis of Nucleotide from PRPP  PRPP is converted to a nucleotide by addition of a base  reaction is reversible, but it proceeds almost exclusively to the right  PPi product is readily cleaved by pyrophosphatases Guanine + Guanine + PPipurine metabolism 16
  16. 16. Purine Metabolism guanosine synthesis | degradation | clinical disorders
  17. 17. PurineNucleotideSynthesis:De NovoPathway 18
  18. 18. Purine Nucleotide Synthesis: De Novo Pathway purine ring: utilizes amino acids (glutamine, glycine, aspartate) as carbon and nitrogen donors, tetrahydrofolate as a one-carbon donor, and CO2 as a carbon donor 19
  19. 19. Purine Nucleotide Synthesis: De Novo Pathway consists of 10 metabolic steps  inosine 5’-monophosphate (IMP) IMP – first ribonucleotide formed; precursor for both AMP and GMP utilizes PRPP as an important substrate ATP needed to drive several reactions of the pathway (6 moles of ATP) present in most cells, not all (e.g. RBCs) highly-regulated process 20
  20. 20. Purine Nucleotide Synthesis: De Novo Pathway  formation of 5- phosphoribosylamine (first step) by glutamine PRPP amidotransferase, is the committed and regulated step  the N-C bond formed will become the N- glycosidic bond of the purine nucleotide  IMP 21
  21. 21. Purine Nucleotide Synthesis: De Novo Pathway IMP - common precursor for AMP and GMP 22
  22. 22. Formation of GMP from IMP xanthosine 5‟-monophosphate (XMP) is formed by the oxidation of IMP catalyzed by IMP dehydrogenase guanosine 5‟monophosphate (GMP) is formed by the addition of an amino group from glutamine to XMP catalyzed by GMP synthetase 23
  23. 23. Formation of AMP from IMP adenylosuccinate is formed by the addition of aspartate to IMP catalyzed by adenylosuccinate synthetase adenosine 5‟-monophosphate (AMP) is formed by the cleavage of fumarate from adenylosuccinate by adenylosuccinase 24
  24. 24. Formation of GMP and AMP from IMP  IMP  GMP : requires ATP as energy source  IMP  AMP: requires GTP as energy source  reciprocal relationship ensures that when there is sufficient ATP in the cell, GMP will be synthesized from IMP and when there is sufficient GTP, AMP will be synthesized from IMPregulation 25
  25. 25. De Novo Purine Nucleotide Synthesis Regulation  formation of 5- phosphoribosylamine (PRA) is the committed step in IMP formation  glutamine PRPP- amidotransferase - rate-limiting; regulated allosterically by the substrates and end products of the pathway 26
  26. 26. De Novo Purine Nucleotide Synthesis Regulation glutamine PRPP-amidotransferase with  IMP, GMP or AMP: the enzyme is dimeric  inactive IMP, GMP and AMP: negative effectors with  PRPP: the enzyme is monomeric  active PRPP: positive effector 27
  27. 27. De Novo Purine Nucleotide Synthesis Regulation Other points of regulation:  branch point of IMP to GMP: IMP Dehydrogenase (IMPDH*) is rate-limiting enzyme and is regulated by GMP acting as a competitive inhibitor of IMDPH  branch point of IMP to * ** AMP: adenylosuccinate synthetase** is rate limiting in conversion of IMP to AMP, with AMP acting as competitive inhibitorsalvage 28
  28. 28. Purine Nucleotide Synthesis: Salvage Pathway two specific enzyme systems (phosphoribosyl transferases) catalyze the transfer of the ribose phosphate from PRPP to free purine bases 29
  29. 29. Purine Nucleotide Synthesis: Salvage Pathway Hypoxanthine-guanine phosphoribosyl transferase (HGPRTase)  catalyzes the formation of nucleotides from either hypoxanthine or guanine  hypoxanthine and guanine for salvage arise from degradation of purine nucleotides  IMP and GMP are competitive inhibitors of PRPP for HGPRTase 30
  30. 30. Purine Nucleotide Synthesis: Salvage Pathway Adenine phosphoribosyl transferase (APRTase)  catalyzes the formation of AMP from adenine  AMP is a competitive inhibitor of PRPP for APRTaseeffects 31
  31. 31. Effects of Salvage on De No Synthesis Pathway  salvage pathway synthesis of purine nucleotides affectively decreases the de novo pathway at the PRPP amidotransferase step:  PRPP is consumed  decreasing the rate of formation of 5-phosphoribosylamine (rate limiting step of de novo pathway)  AMP, IMP and GMP serve as feedback inhibitors at this step  salvage reactions conserve energy and permit cells to form nucleotides from free nucleobases or nucleosides  helpful in cells without glutamine PRPP amidotransferase (e.g., RBCs)interconversions 32
  32. 32. Purine Nucleotide Interconversions  occur by indirect steps; no direct one-step pathway for conversion of GMP to AMP or AMP to GMP  AMP or GMP is converted to IMPpurine degradation 33
  33. 33. PurineNucleotideDegradation  degradation of purine nucleotides, nucleosides and nucleobases follow a common pathway that leads to the formation of uric acid 34
  34. 34. adenine nucleotides  hypoxanthine guanine nucleotides  xanthineUA 35
  35. 35. Uric Acid Formation  hypoxanthine and xanthine - oxidized by xanthine oxidoreductase (XOR), which has both dehydrogenase and oxidase activities  xanthine dehydrogenase activity requires NAD+ as the electron acceptor  xanthine oxidase activity utilizes molecular oxygen with the generation of hydrogen peroxide  uric acid is the unique end product of purine nucleotide degradation in man 36
  36. 36. Uric Acid  final oxidation product of purine metabolism  a very weak organic acid that is barely soluble in water and insoluble in alcohol and ether  urates are its saltsd/o 37
  37. 37. Clinical Disordersof Purine Metabolism
  38. 38. Hyperuricemia and Gout  Gout - manifestation of acute or prolonged hyperuricemia  precipitation of blood urate as crystals of sodium urate in the synovial fluid of joints  inflammation painful arthritis, which if untreated, leads ultimately to severe degeneration of the joints  causesmanifestations 39
  39. 39. Gout: Manifestationscauses 40
  40. 40. Gout: Causes APRTase PRPP PRPP synthetase amido transferase 1 2 Elevated Loss of levels feedback HGPRTase Decreased inhibition Levels 3 41
  41. 41. Gout: Causes Elevated activity of PRPP Synthetase (defect 1)  insensitivity to feedback inhibition by purine nucleotides   PRPP   activity of PRPP amidotransferase reaction (rate-limiting step in de novo purine synthesis)   synthesis of purines and uric acid 42
  42. 42. Gout: Causes Mutations in PRPP amidotransferase (defect 2)  makes the enzyme less sensitive to feedback inhibition by purine nucleotides   loss of control also increases flux through the rate-limiting step   synthesis of purines and uric acid 43
  43. 43. HGPRTase Deficiency Gout: Causes  (defect 3)  HGPRTase reaction, when active, consumes PRPP  Decreased flux through this reaction, when HGPRTase is deficient   steady state level of PRPP;  IMP and GMP   intracellular activity of PRPP amidotransferase   synthesis of purines and uric acidother causes 44
  44. 44. Gout: Other Causes  impaired uric acid excretion (renal pathology)  increase rate of nucleic acids degradation (cancer chemotherapy / radiotherapy)tx 45
  45. 45. Hyperuricemia and Gout: Treatment  dietary – decrease intake of purine rich-foods  pharmacologic – Allopurinol  structural analog of hypoxanthine that strongly inhibits xanthine oxidoreductase  inhibition causes accumulation of hypoxanthine and xanthine, both of which are more soluble and more excretable than uric acidLNS 46
  46. 46. Lesch-Nyhan Syndrome (LNS / LND)  a rare, inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) (defect 3)  increase in intracellular PRPP results in purine overproduction  manifests as severe gouty arthritis and some neurologic manifestations: behavioral disorders, learning disability, hostile and aggressive behaviors towards self (self-mutilation) 47
  47. 47. Von Gierke’s Disease glucose-6-phosphatase deficiency - increased availability of G6P for the pentose phosphate pathway   production of the PRPP precursor, ribose-5-phosphate   PRPP  PRPP accelerates de novo purine synthesis leading to purine overproduction and hyperuricemia associated lactic acidosis elevates the renal threshold for urate, elevating total body urates 48
  48. 48. Hypouricemia associated with increased excretion of hypoxanthine and xanthine caused by deficiency of xanthine oxidoreductase due to a genetic defect or to severe liver damage patients may exhibit xanthinuria and xanthine lithiasis 49
  49. 49. Adenosine Deaminase (ADA) Deficiency  Severe Combined Immunodeficiency Syndrome (SCID) - both T-cells and B-cells are deficient and dysfunctional   deoxyadenosine and adenosine   deoxyATP which“Bubble Boy Disease” inhibit ribonucleotide reductase resulting to inhibition of DNA synthesis and less proliferation of lymphocytes  recurrent or chronic infections, failure to thrive, fatal before 18 years of age 50
  50. 50. What Would They Say (Paul Williams) What would they say If we up and ran away From the roaring crowds And the worn out city faces Would they carry on and on When they found out we were gone Or would they let us go Would they tag along Or would they know to What would they do If they found out we were through With all the little lies And the downtown aggravations That wed traded them away For a quiet country day That we had hoped to share Would they try to find out Where we were or What would they say If we up and ran away Ran away 51
  51. 51. Adenosine Deaminase (ADA) Deficiency  DNA  lymphocytes synthesis 52
  52. 52. Purine Nucleoside Phosphorylase (PNP) Deficiency  less severe than ADA deficiency  associated with severe deficiency of T-cells but apparently normal B-cell function  immune dysfunctions appear to result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase and thereby deplete cells of DNA precursors 53
  53. 53. Purine Nucleoside Phosphorylase (PNP) Deficiency  T-cell lymphocytes 54
  54. 54. Pyrimidine Metabolism uridine synthesis | degradation | clinical disorders
  55. 55. Pyrimidine NucleotideSynthesis: De Novo Pathway  de novo synthesis of the pyrimidine ring in mammalian cells utilizes amino acids and nitrogen donors in addition to CO2.  Uridine 5‟- monophosphate (UMP) is synthesized in a six- step metabolic pathway, requiring ATP hydrolysis 56
  56. 56. Pyrimidine Nucleotide Synthesis: De Novo Pathway requires aspartate as carbon and nitrogen donor, glutamine as a nitrogen donor and CO2 as a carbon donor five of the six reactions occur in the cytosol of the cell, while the other reaction occurs in the mitochondria 57
  57. 57. Pyrimidine Synthesis pyrimidine ring is formed first; ribose 5-phosphate is added with PRPP as the ribose 5-phosphate donor formation of cytosolic carbamoyl phosphate (CPS II) is the regulated step formation of N- carbamoylaspartate is the committed step in pyrimidine nucleotide synthesis 58
  58. 58. Pyrimidine Synthesis  nucleotide kinases convert UMP to UTP  UTP serves as substrate for CTP synthetase 59
  59. 59. Formation of CTP from UTP CTP synthetase catalyzes the formation of CTP from UTP with glutamine as the amino group donor the product, CTP is a negative effector of CTP synthetase this regulation maintains an appropriate ratio of UTP and CTP for cellular function and RNA synthesis 60
  60. 60. Pyrimidine NucleotideSynthesis Regulation regulation of pyrimidine nucleotide synthesis occurs at the CPS II step CPS II is inhibited by CTP, an end product of the pathway CPS II is activated by PRPPsalvage pathway 61
  61. 61. Pyrimidine Nucleotide Synthesis: Salvage Pathway  pyrimidine bases are „salvaged‟ to reform nucleotides by pyrimidine phosphoribosyl transferase: pyrimidine + PRPP  pyrimidine nucleoside 5’-monophosphate + PPi  these reactions divert the pyrimidine bases from the degradative pathway to nucleotide formation  as a pyrimidine base becomes available to cells, there are competing reactions that will result in either: a. degradation and excretion of the products b. reutilization of the bases for nucleotide synthesisdegradation 62
  62. 62. PyrimidineNucleotideDegradation 63
  63. 63. Degradation of Pyrimidine Nucleobases uracil and thymine are degraded by analogous reactions uracil is degraded to -alanine, NH4+ and CO2 thymine is degraded to -aminoisobutyric acid, NH4+ and CO2. 64
  64. 64. Clinical Disorders of Pyrimidine Metabolism Orotic Acidurias  Reye Syndrome: consequence of the inability of severely damaged mitochondria to utilize carbamoyl phosphate, which then becomes available for cytosolic overproduction of orotic acid  deficiency of liver mitochondrial ornithine transcarbamoylase (an enzyme in the urea cycle)   excretion of orotic acid, uracil and uridine  excess carbamoyl phosphate exits from the mitochondria to the cytosol, where it stimulates pyrimidine nucleotide biosynthesisdntp 65
  65. 65. DeoxyribonucleotideMetabolism thymidine synthesis | interconversions
  66. 66. Deoxyribonucleotide Biosynthesis synthesis of deoxyribonucleoside triphosphates (dNTPs) is important in DNA synthesis and repair nucleoside 5’-diphosphate reductase (ribonucleotide reductase) – catalyzes the reaction in which ribonucleoside 5‟-diphosphates are reduced to 2‟-deoxyribonucleoside 5‟- diphosphates 67
  67. 67. Deoxyribonucleotide Biosynthesis  nucleoside 5’-diphosphate reductase (ribonucleotide reductase) – catalyzes the reaction in which ribonucleoside 5‟- diphosphates are converted to 2‟- deoxyribonucleoside 5‟-diphosphatesregulation 68
  68. 68. Deoxyribonucleotide Biosynthesis: Regulation reaction is controlled by the amount of enzyme present in cells and by a very finely-regulated allosteric control mechanism (NTPs) thioredoxin or glutaredoxin: involved in reduction at the 2‟-position through oxidation of its sulfhydryl groups NADPH is used to regenerate free sulfhydryl groups on thioredoxin or glutaredoxin 69
  69. 69. Deoxyribonucleotide Biosynthesis: Regulation reduction of a particular substrate requires a specific nucleoside 5‟-triphosphate as a positive effector Major Positive Substrate Major Negative Effector Effector CDP ATP dATP, dGTP, dTTP UDP ATP dATP, dGTP, dTTP ADP dGTP dATP GDP dTTP dATP 70
  70. 70. Role of Ribonucleotide Reductase in DNA Synthesis ribonucleotide reductase (1) catalyzes the rate-limiting reactions in which 2‟-deoxyribonucleoside 5‟-triphosphates are synthesized de novo for DNA replication inhibitors of ribonucleotide reductase are potent inhibitors of DNA synthesis and, hence, of cell replication 71
  71. 71. DeoxythymidylateBiosynthesis thymidylate synthase catalyzes the transfer of a one-carbon unit from N5,N10-methyleneH4folate to dUMP and which is simultaneously reduced to a methyl group N5,N10-methyleneH4folate serves as a one-carbon donor and as a reducing agent 72
  72. 72. Deoxyribonucleotide Interconversions dCTP and dTTP are major positive and negative effectors of the interconversions and salvage of deoxyribonucleosidesRx 73
  73. 73. Pharmacologic Agentsthat Interfere withNucleotide Metabolism AZT (Zidovudine) antimetabolites | antifolates | glutamine antagonists | antivirals | other agents
  74. 74. Pharmacologic Agents  purine and pyrimidine nucleotides: critical for normal cell function, maintenance and replication  compounds may be specific inhibitors of enzymes involved in nucleotide synthesis, inter- conversions or degradation  antimetabolites, antifolates. glutamine antagonists, other agentsantimetabolites 75
  75. 75. Antimetabolites structural analogs of purine and pyrimidine bases or nucleosides that interfere with very specific metabolic reactions  6-Mercaptopurine (6-MP)  5-Fluorouracil (Fura or 5-FU)  Cytosine Arabinoside (AraC) 76
  76. 76. Antimetabolites: 6-Mercaptopurine (6-MP)  useful antitumor drug in humans  utilizing PRPP and HGPRTase, 6-mercaptopurine ribonucleoside 5‟- monophosphate is formed in cells and serves as a negative effector of PRPP-amidotransferase  this nucleotide also act as an inhibitor of the conversion of IMP to GMP at the IMP-dehydrogenase step and IMP to AMP at the adenylosuccinate synthetase step 77
  77. 77. Antimetabolites: 5-Fluorouracil (5-FU)  analog of uracil  converted to the active metabolite 5- fluorouridine 5‟-triphosphate (FUTP) and 5-fluoro-2‟-deoxyuridine 5‟- monophosphate (FdUMP).  FUTP is efficiently incorporated into RNA  inhibits its maturation  FdUMP is a potent and specific irreversible inhibitor of thymidylate synthase, resulting in the inhibition of dTMP synthesis and leads to “thymineless death” for cells 78
  78. 78. Antimetabolites: Cytosine Arabinoside (AraC)  treatment of several forms of cancer (leukemias)  metabolized to cytosine arabinoside 5‟-triphosphate (araCTP) to exert its cytotoxic effects  araCTP competes with dCTP in the DNA polymerase reaction and araCMP is incorporated into DNA, which inhibits the synthesis of the growing strandantifolates 79
  79. 79. Antifolates  folate analogs interfere with metabolic steps in which tetrahydrofolate is involved as either as a substrate or product  purine de novo synthesis  thymidylate synthase (dUMP  dTMP) 80
  80. 80. Antifolates: Methotrexate (MTX) synthetic analog of folic acid  amino group at C4 instead of OH  methyl group at N10 instead of an H 81
  81. 81. Antifolates:Methotrexate (MTX) interferes with the formation of H2folate and H4folate from folate by specifically inhibiting H2folate reductase (DHFR). 82
  82. 82. Antifolates: Methotrexate (MTX)  cells would be incapable of de novo synthesis of purine nucleotides or thymidylate synthesis due to depletion of H4folate pools   lowering of ribonucleoside 5‟- triphosphate and 2‟-deoxyribo- nucleoside 5‟- triphosphate “thymineless death” intracellular poolsGA 83
  83. 83. Glutamine Antagonists  many reactions in the cells utilize glutamine as the amino group donor  amidation reactions are critical:  de novo synthesis of purine nucleotide (N3 and N9)  synthesis of GMP from IMP  formation of cytosolic carbamoyl phosphate  synthesis of CTP from UTP  synthesis of NAD+.  compounds that inhibit these reactions are referred to as glutamine antagonists 84
  84. 84. Glutamine Antagonists:O-diazoacetyl-L-serine (Azaserine)6-diazo-5-oxo-L-norleucine (DON)  effective inhibitors of enzymes that utilize glutamine as the amino donor  addition of glutamine alone will not reverse the effects of these two drugs  many metabolites (guanine, cytosine, hypoxanthine/adenine and nicotinamide): needed to bypass the many sites blocked by these glutamine antagonists(DON) Azaserine AV 85
  85. 85. Antivirals enzymes of nucleotide synthesis have been widely studied as target sites for the action of antiviral or antimicrobial drugs purine and pyrimidine analogs can target these enzymes and thus control or help treat certain viral diseases 86
  86. 86. Antivirals: Acylguanosine (Acyclovir)  purine analog, commonly used in the treatment of herpesvirus (HSV)  converted to the monophosphate by a specific HSV-thymidine kinase present only in virally-infected cells  acycloguanosine monophosphate is phosphorylated to the di- and triphosphate forms  acycloguanosine triphosphate serves as a substrate for the HSV- specific DNA polymerase  incorporated in the growing DNA chain, causing chain termination 87
  87. 87. Antivirals: 3’-azido-3’deoxythymidine (AZT)  pyrimidine analog, commonly used in the treatment of human immunodeficiency virus (HIV) infections  phosphorylated by cellular kinases to AZT- triphosphate, which blocks HIV replication by inhibiting HIV-DNA polymerase OA 88
  88. 88. Other Agents: Hydroxyurea  limited use in cancer treatment; also used in the treatment of sickle cell anemia  inhibits ribonucleotide reductase activity by destroying the tyrosyl free radical on the small subunit of the enzyme  inhibition of the reduction of CDP, UDP, GDP and ADP to the corresponding 2‟- deoxyribonucleoside 5‟- diphosphates  termination of DNA replication 89
  89. 89. Other Agents: Tiazofurin  converted by cellular enzymes to the NAD+ analog tiazofurin adenine dinucleotide (TAD)  TAD inhibits IMP dehydrogenase, the rate-limiting enzyme in GTP synthesis   GTP   dGTP 90
  90. 90. Thank you very much! adenosine triphosphate Noel Martin S. Bautista, MD, MBAH Department of Biochemistry, Molecular Biology and Nutrition

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