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Nucleotide metabolism – Part 1 (purine biosynthesis). By Henry Wormser, Ph.D Professor of Medicinal Chemistry. Biological significance of nucleotide metabolism. Nucleotides make up nucleic acids (DNA and RNA) Nucleotide triphosphates are the “energy carriers” in cells (primarily ATP)

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nucleotide metabolism part 1 purine biosynthesis

Nucleotide metabolism – Part 1(purine biosynthesis)

By

Henry Wormser, Ph.D

Professor of Medicinal Chemistry

biological significance of nucleotide metabolism
Biological significance of nucleotide metabolism
  • Nucleotides make up nucleic acids (DNA and RNA)
  • Nucleotide triphosphates are the “energy carriers” in cells (primarily ATP)
  • Many metabolic pathways are regulated by the level of the individual nucleotides
    • Example: cAMP regulation of glucose release
  • Adenine nucleotides are components of many of the coenzymes
    • Examples: NAD+, NADP+, FAD, FMN, coenzyme A
dietary nucleotides
Dietary nucleotides
  • do not contribute energy as do carbs, proteins and fats
  • are not incorporated into RNA or DNA unless given I.V.
  • normally metabolized to individual components (bases, sugar and phosphate)
  • purines are converted to uric acid which is then excreted
medical significance of nucleotide metabolism
Medical significance of nucleotide metabolism
  • Anticancer agents:
      • Rapidly dividing cells biosynthesize lots of purines and pyrimidines, but other cells reuse them. Cancer cells are rapidly dividing, so inhibitor of nucleotide metabolism kill them
  • Antiviral agents
    • Zidovudine (Retrovir)
    • Lamivudine (Epivir)
    • Valacyclovir (Valtrex)
structures of nucleotide building blocks and nucleotides7
Structures of nucleotide buildingblocks and nucleotides

guanine: comes from guano; thymine –thymus gland

synthesis of inosine monophosphate
Synthesis of Inosine Monophosphate
  • Basic pathway for biosynthesis of purine ribonucleotides
  • Starts from ribose-5-phosphate which is derived from the pentose phosphate pathway
  • Requires 11 steps overall
  • occurs primarily in the liver
steps 1 thru 3
Steps 1 thru 3
  • Step 1:Activation of ribose-5-phosphate
    • enzyme: ribose phosphate pyrophosphokinase
    • product: 5-phosphoribosyl-a-pyrophosphate (PRPP)
    • PRPP is also a precursor in the biosynthesis of pyrimidine nucleotides and the amino acids histidine and tryptophan
steps 1 thru 314
Steps 1 thru 3
  • Step 2: acquisition of purine atom 9
    • enzyme: amidophosphoribosyl transferase
    • displacement of pyrophosphate group by glutamine amide nitrogen (inversion of configuration – a to b
    • product: b-5-phosphoribosylamine

Steps 1 and 2 are tightly regulated by feedback inhibition

steps 1 thru 316
Steps 1 thru 3
  • Step 3: acquisition of purine atoms C4, C5, and N7
    • enzyme: glycinamide synthetase
    • b-phosphoribosylamine reacts with ATP and glycine
    • product: glycinamide ribotide (GAR)
steps 4 thru 6
Steps 4 thru 6
  • Step 4: acquisition of purine atom C8
    • formylation of free a-amino group of GAR
    • enzyme: GAR transformylase
    • co-factor of enzyme is N10-formyl THF
  • Step 5: acquisition of purine atom N3
    • The amide amino group of a second glutamine is transferred to form formylglycinamidine ribotide (FGAM)
  • Step 6: closing of the imidazole ring or formation of 5-aminoimidazole ribotide
step 7
Step 7
  • Step 7: acquisition of C6
    • C6 is introduced as HCO3-
    • enzyme: AIR carboxylase (aminoimidazole ribotide carboxylase)
    • product: CAIR (carboxyaminoimidazole ribotide)
    • enzyme composed of 2 proteins: PurE and PurK (synergistic proteins)
steps 8 thru 11
Steps 8 thru 11
  • Step 8: acquisition of N1
    • N1 is acquired from aspartate in an amide condensation reaction
    • enzyme: SAICAR synthetase
    • product: 5-aminoimidazole-4-(N-succinylocarboxamide)ribotide (SAICAR)
    • reaction is driven by hydrolysis of ATP
steps 8 thru 1125
Steps 8 thru 11
  • Step 9: elimination of fumarate
    • Enzyme: adenylosuccinate lyase
    • Product: 5-aminoimidazole-4-carboxamide ribotide (AICAR)
  • Step 10: acquisition of C2
    • Another formylation reaction catalyzed by AICAR transformylase
    • Product: 5-formaminoimidazole-4-carboxamide ribotide (FAICAR)
step 11
Step 11
  • cyclization or ring closure to form IMP
  • water is eliminated
  • in contrast to step 6 (closure of the imidazole ring), this reaction does not require ATP hydrolysis
  • once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells
slide31

Synthesis of adenine

and guanine nucleotides

slide32

Purine nucleoside diphosphates and triphosphates:

- to be incorporated into DNA and RNA, nucleoside

monophosphates (NMP’s) must be converted into

nucleoside triphosphates (NTP’s)

- nucleoside monophosphate kinases (adenylate & guanylate kinases)

- nucleoside diphosphate kinase

the purine salvage pathway
The purine salvage pathway
  • Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis were costly for the cell to make, so there are pathways to recover these bases in the form of nucleotides
  • Two phosphoribosyl transferases are involved:
    • APRT (adenine phosphoribosyl transferase) for adenine
    • HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine
salvage of purines
Salvage of purines

Adenine phosphoribosyltransferase (APRT)

salvage of purines37
Salvage is needed to maintain the purine pool (biosynthesis is not completely adequate, especially in neural tissue)

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

Hypoxanthine + PRPP IMP + Ppi

Guanine + PRPP GMP + Ppi

Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of enzyme leads to overproduction of purines which are metabolized to uric acid, which damages cells

Salvage of purines
lesch nyhan syndrome
Lesch-Nyhan syndrome
  • there is a defect or lack in the HGPRT enzyme
  • the rate of purine synthesis is increased about 200X
  • uric acid level rises and there is gout
  • in addition there are mental aberrations
  • patients will self-mutilate by biting lips and fingers off
nucleotide metabolism part 2 pyrimidine biosynthesis

Nucleotide metabolism – Part 2(pyrimidine biosynthesis)

By

Henry Wormser, Ph.D

Professor of Medicinal Chemistry

synthesis of pyrimidine ribonucleotides
Synthesis of pyrimidine ribonucleotides
  • shorter pathway than for purines
  • base is made first, then attached to ribose-P (unlike purine biosynthesis)
  • only 2 precursors (aspartate and glutamine, plus HCO3-) contribute to the 6-membered ring
  • requires 6 steps (instead of 11 for purine)
  • the product is UMP (uridine monophosphate)
step 1 synthesis of carbamoyl phosphate
Step 1: synthesis of carbamoyl phosphate
  • Condensation of glutamine, bicarbonate in the presence of ATP
  • Carbamoyl phosphate synthetase exists in 2 types: CPS-I which is a mitochondrial enzyme and is dedicated to the urea cycle and arginine biosynthesis) and CPS-II, a cytosolic enzyme used here
step 1 pyrimidine synthesis
Step 1: pyrimidine synthesis

CPS-II is the major site of regulation in animals: UDP and

UTP inhibit the enzyme and ATP and PRPP activate it

It is the committed step in animals

step 2 synthesis of carbamoyl aspartate
Step 2: synthesis of carbamoyl aspartate
  • enzyme is aspartate transcarbamoylase (ATCase)
  • catalyzes the condensation of carbamoyl phosphate with aspartate with the release of Pi
  • ATCase is the major site of regulation in bacteria; it is activated by ATP and inhibited by CTP
  • carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step
step 3 ring closure to form dihydroorotate
Step 3: ring closure to form dihydroorotate
  • enzyme: dihydroorotase
  • forms a pyrimidine from carbamoyl aspartate
  • water is released in this process
slide52
the first 3 enzymatic reactions are catalyzed by 3 separate proteins/enzymes in E. coli
  • in animals, all 3 steps are found in a multifunctional enzyme (210 kD). This allows “channeling” of the substrates and products between active sites without releasing them to the medium where they could be degraded.
  • The acronym CAD is used as a name for the multienzyme: carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase
  • channeling also increases the overall rate of multistep processes
step 4 oxidation of dihydroorotate to orotate
Step 4: oxidation of dihydroorotate to orotate
  • an irreversible reaction
  • enzyme: dihydroorotate dehydrogenase
  • oxidizing power is derived from quinones (thru coenzyme Q)
step 5 acquisition of ribose phosphate moiety
Step 5: acquisition of ribose phosphate moiety
  • enzyme: orotate phosphoribosyl transferase
  • ribose phosphate originates from PRPP
  • product is orotidine-5’-monophosphate (OMP)
  • orotate phosphoribosyl transferase is also used in salvage of uracil and cytosine to their corresponding nucleotide
step 6 decarboxylation of omp
Step 6: decarboxylation of OMP
  • enzyme: OMP decarboxylase
  • product: uridine monophosphate (UMP)
  • in animals, steps 5 and 6 are catalyzed by a single polypeptide with 2 active sites
orotic aciduria
Orotic aciduria
  • an inherited human disease caused by a deficiency in the multifunctional enzyme that catalyzes the last 2 steps in the pyrimidine synthesis
  • large amounts of orotic acid in urine
  • retarded growth and severe anemia
  • treat by administration (injection) of uridine and/or cytidine
leflunomide arava
Leflunomide (Arava)
  • Leflunomide is an isoxazole immunomodulatory agent which inhibits dihydroorotate dehydrogenase) and has antiproliferative activity. Several in vivo and in vitro experimental models have demonstrated an anti-inflammatory effect.
  • It is currently used as a DMARD in patients with serious rheumatoid arthritis
activation of leflunomide
Activation of leflunomide

Opening of the isoxazole yields a reactive compound which

can then inhibit the enzyme dihydroorotate dehydrogenase

synthesis of uridine and cytidine triphosphate
Synthesis of uridine and cytidine triphosphate

(in bacteria, ammonia donates the amino group)

regulation of pyrimidine nucleotide biosynthesis
Regulation of pyrimidine nucleotide biosynthesis

UTP and CTP are feeback inhibitors of CPS II

formation of deoxyribonucleotides
Formation of deoxyribonucleotides

All pathways shown previously led to synthesis of ribonucleotides

dADP, dGDP, dUDP and dCDP are all synthesized by the same enzyme

Synthesized from nucleoside diphosphate (not mono or triphosphate) by

ribonucleotide reductase

synthesis of dtmp
Synthesis of dTMP
  • Methylation of d-UMP via N5,N10-methylene THF
  • Reaction inhibited by 5-fluorouracil (Efudex)
nucleotide metabolism part 3 nucleotide degradation

Nucleotide metabolism – Part 3(nucleotide degradation)

By

Henry Wormser, Ph.D

Professor of Medicinal Chemistry

pentostatin
PENTOSTATIN

previously called deoxycoformycin (DCF)

a purine analog with a 7-membered-ring

potent inhibitor of adenosine deaminase

ADA is a key enzyme which regulates

adenosine levels in cells

indicated for refractory hairy cell leukemia

other uses: chronic lymphocytic leukemia

and lymphomas

ada deficiency
ADA deficiency
  • In the absence of ADA lymphocytes are destroyed
  • deoxyadenosine is not destroyed, is converted to dAMP and then into dATP
  • dATP is a potent feedback inhibitor of deoxynucleotide biosynthesis
  • this leads to SCID (severe combined immunodeficiency disease)
  • Infants with this deficiency have a high fatality rate due to infections
ada deficiency84
ADA deficiency
  • treatment consists of administering pegylated ADA which can remain in the blood for 1 – 2 weeks
  • more efficient is gene therapy: replacing the gene that is missing or defective
  • gene therapy has been performed on selected patients
slide85

Degradation of

GMP and XMP

slide89
GOUT
  • a disorder associated with abnormal amounts of urates in the body
  • early stage: recurring acute non-articular arthritis
  • late stage: chronic deforming polyarthritis and eventual renal complication
  • disease with rich history dating back to ancient Greece
slide90
GOUT
  • once fashionable to associate gout with intelligence
  • people with gout:
    • Isaac Newton
    • Benjamin Frankin
    • Martin Luther
    • Charles Darwin
    • Samuel Johnson
slide91
Gout
  • prevails mainly in adult males
  • rarely encountered in premenopausal women
  • symptoms are cause by deposition of crystals of monosodium urate monohydrate (can be seen under polarized light)
  • usually affect joints in the lower extremities (the big toe is the classic site)
four stages of gout
Four Stages of Gout

1. asymptomatic hyperuricemia

2. acute gouty arthritic attacks

3. asymptomatic intercritical period

4. tophaceous gout (characterized by the formation of tophi in joints)

  • podagra (big toe)
  • cheiagra (wrist) according to Hippocrates
  • gonadra (knee)
diagnostic features
Diagnostic features
  • usually affect joints in the lower extremities ( 95%)
  • onset is fast and sudden
  • pain is usually severe; joint may be swollen, red and hot
  • attack may be accompanied by fever, leukocytosis and an elevated ESR
drugs which may induce hyperuricemia
Drugs which may induce hyperuricemia
  • niacin
  • thiazides and other diuretics
  • low dose aspirin
  • pyrazinamide
  • ethambutol
  • cyclosporine
  • cytotoxic drugs
non pharmacological approaches
Non-pharmacological approaches
  • Avoid purine rich foods:
    • red meat and organ meat (liver, kidneys)
    • shellfish, anchovies, mackerel, herring
    • meat extracts and gravies
    • peas and beans, aspargus, lentils
    • beer, lager, other alcoholic beverages
  • Weight loss
  • Control alcohol (binge drinking)
pharmacological management of gout
Pharmacological management of gout
  • based on the premise that the hyperuricemia is due to both overproduction and underexcretion of uric acid
  • symptomatic relief of pain is also achieved with analgesics (i.e. indomethacin)
  • drugs used:
    • analgesics (NSAIDs)
    • uricosuric agents
    • xanthine oxidase inhibitors
therapy of acute gout
Therapy of acute gout
  • treat with colchicine or NSAIDs
  • avoid aspirin
  • do not treat with allopurinol or uricosuric drugs
  • uric acid lowering agents should never be started or stopped during acute attack
  • pain resolution occurs within 48-72 hrs
colchicine
Colchicine

a non-basic alkaloid from the seeds and corms of Colchicum autumnale

(Meadow Safron)

colchicine100
COLCHICINE
  • used in the symptomatic treatment of acute attacks of gout
  • decreases leukocyte motility, decreases phagocytosis and lactic acid production
  • not used in other forms of arthritis
  • a very potent drug
  • can cause severe GI distress and abdominal pain
probenecid benemid
Probenecid (Benemid)

A uricosuric agent

probenecid benemid102
Probenecid (Benemid)
  • inhibits the tubular reabsorption of uric acid
  • it can also inhibit the tubular excretion of certain organic acid via the transporter
  • used in gout to promote the elimination of uric acid (not effective in acute attack)
  • also used to enhance plasma concentration of certain antiinfectives (beta lactams)
allopurinol zyloprim
ALLOPURINOL (Zyloprim)
  • prevention of attacks of gouty arthitis and nephropathy
  • also used during chemotherapy of cancer and to prevent recurrent calcium oxalate calculi
  • metabolized to oxypurinol (also an inhibitor of xanthine oxidase)
  • inhibits the metabolism of certain anticancer drugs (6-MP, azathioprine)
allopurinol zyloprim104
Allopurinol (Zyloprim)

An inhibitor of xanthine oxidase; prevents the formation of uric acid from

precursorial purines

fate of uric acid
Fate of uric acid
  • in human and other primates uric acid is the final product of purine degradation and is excreted in the urine
  • the same is true in bird, reptiles and many insects
  • in other mammals uric acid is oxidized to allantoin (urate oxidase)
  • teleost (bony) fish convert allantoin to allantoic acid
  • cartilaginous fish and amphibian further degrade allantoic acid to urea
  • and finally marine invertebrates decompose urea to ammonia
rasburicase elitek
Rasburicase (Elitek)

A recombinant form of uric

acid oxidase. Used for initial management of plasma uric acid levels in pediatric patients with leukemia, lymphoma, and solid tumor malignancies who are receiving anticancer therapy expected to result in tumor lysis and subsequent elevation of plasma uric acid.

formation of deoxyribonucleotides112
Formation of deoxyribonucleotides
  • ribonucleotide reductase studied by JoAnne Stubbe (Wisconsin, then MIT)
  • very complex enzyme; contains:
      • Tyrosine radical
      • 2 non-heme irons
      • Two catalytically active cysteine residues
      • Cys are reduced by other proteins – thioredoxin
      • Ribo. Reductase is the therapeutic target of the anticancer drug hydroxyurea
mechanism of ribonucleotide reductase
Mechanism of ribonucleotide reductase
  • Free radical mechanism involving tyrosyl residues and cysteine residues on the enzyme
  • The enzyme is a dimer of dimers:
    • R1 – a dimer of identical a subunits (85 kD each)
    • R2 – a dimer of identical b subunits (45 kD each)
reduction of the disulfide bond in ribonucleotide reductase
Reduction of the disulfide bond in ribonucleotide reductase
  • 2 proteins can perform this reductive reaction:
    • Thioredoxin (ubiquitous 12 kD monomer)
    • Glutaredoxin which functions similarly to thioredoxin. Oxidized glutredoxin is reduced by glutathione (g-glutamylcysteinylglycine)
hydroxyurea hydrea
HYDROXYUREA (Hydrea)
  • inhibits the enzyme ribonucleotide reductase
    • this enzyme causes ribonucleotides to be converted to deoxyribonucleotides
    • DNA synthesis cannot occur
    • cell are killed in the S phase
    • drug holds other cells in the G1 phase
  • primarily used to treat chronic myelogenous leukemia
  • cancer cell develop resistance by:
    • increasing quantity of inhibited enzyme
    • decreasing sensitivity of enzyme for inhibitor
  • used orally
  • major side effect is leukopenia
gemcitabine gemzar
GEMCITABINE (Gemzar)

Another inhibitor of ribonucleotide reductase:indicated for non-small cell lung cancer (usually with cisplatin) also first line treatment for non-resectable pancreatic cancer

slide123

The purine nucleotide cycle for anaplerotic replenishment of citric acid cycle intermediates in skeletal muscle

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