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Nucleotides:Nucleotides: Synthesis and Synthesis and DegradationDegradation
Javad Zavar RezaPh.D in Clinical Biochemistry Ph.D in Clinical Biochemistry
Department of Biochemistry Department of Biochemistry School of Medicine School of Medicine
11
Nitrogenous BasesNitrogenous Bases
Planar, aromatic, and heterocyclicPlanar, aromatic, and heterocyclic
Derived from Derived from purinepurine or or pyrimidinepyrimidine
Numbering of bases is “unprimed”Numbering of bases is “unprimed”
22
Nucleic Acid BasesNucleic Acid Bases
Purines Pyrimidines
33
SugarsSugars
Pentoses (5-C sugars)Pentoses (5-C sugars)
Numbering of sugars is “primed”Numbering of sugars is “primed”
44
SugarsSugars
D-Ribose and 2’-DeoxyriboseD-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group
55
NucleosidesNucleosides
66
Phosphate GroupsPhosphate Groups
Mono-, di- or triphosphatesMono-, di- or triphosphates
Phosphates can be bonded to either C3 or C5 Phosphates can be bonded to either C3 or C5 atoms of the sugaratoms of the sugar
77
NucleotidesNucleotides
Result from linking one or more phosphates with a Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through nucleoside onto the 5’ end of the molecule through esterificationesterification
88
NucleotidesNucleotides
Monomers for nucleic acid polymersMonomers for nucleic acid polymers
Nucleoside Triphosphates are important Nucleoside Triphosphates are important energy carriers (ATP, GTP)energy carriers (ATP, GTP)
Important components of coenzymesImportant components of coenzymes– FAD, NADFAD, NAD++ and Coenzyme A and Coenzyme A
99
Naming ConventionsNaming Conventions
Nucleosides:Nucleosides:– Purine nucleosides end in “-sine” Purine nucleosides end in “-sine”
Adenosine, GuanosineAdenosine, Guanosine
– Pyrimidine nucleosides end in “-dine”Pyrimidine nucleosides end in “-dine”Thymidine, Cytidine, UridineThymidine, Cytidine, Uridine
Nucleotides:Nucleotides:– Start with the nucleoside name from above and add Start with the nucleoside name from above and add
“mono-”, “di-”, or “triphosphate”“mono-”, “di-”, or “triphosphate”Adenosine Monophosphate, Cytidine Triphosphate, Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine DiphosphateDeoxythymidine Diphosphate
1010
Nucleotide Biosynthesis Nucleotide Biosynthesis
De novo BiosynthesisDe novo Biosynthesis
Salvage BiosynthesisSalvage Biosynthesis
1111
Nucleotide MetabolismNucleotide Metabolism
PURINE RIBONUCLEOTIDES: DPURINE RIBONUCLEOTIDES: De novoe novo– i.e., purines are i.e., purines are notnot initially synthesized as free bases initially synthesized as free bases– First purine derivative formed is Inosine Mono-phosphate First purine derivative formed is Inosine Mono-phosphate
(IMP)(IMP)
1212
1313
Steps 1 Steps 1
Step 1Step 1:Activation of ribose-5-phosphate:Activation of ribose-5-phosphate
– product: 5-phosphoribosyl-a-pyrophosphate product: 5-phosphoribosyl-a-pyrophosphate (PRPP)(PRPP)
– PRPP is also a precursor in the biosynthesis of:PRPP is also a precursor in the biosynthesis of:
pyrimidine nucleotidespyrimidine nucleotides
HistidineHistidine
Tryptophan Tryptophan
1414
Step 1: purine synthesisStep 1: purine synthesis
1515
Step 2: purine synthesis:Step 2: purine synthesis:commited stepcommited step
1616
Step 3 : purine synthesisStep 3 : purine synthesis
1717
1818
Acquisition of purine atom C8 & purine atom N3
Step 6: purine synthesisStep 6: purine synthesis
1919
Step 7: purine synthesisStep 7: purine synthesis
2020
Acquisition of C6 introduced as HCO3-
Steps 8 thru 11Steps 8 thru 11
Step 8Step 8: acquisition of N1: acquisition of N1– N1 is acquired from aspartate in an amide N1 is acquired from aspartate in an amide
condensation reactioncondensation reaction– enzyme: SAICAR synthetaseenzyme: SAICAR synthetase– product: 5-aminoimidazole-4-(N-product: 5-aminoimidazole-4-(N-
succinylocarboxamide)ribotide (SAICAR)succinylocarboxamide)ribotide (SAICAR)– reaction is driven by hydrolysis of ATPreaction is driven by hydrolysis of ATP
2121
Step 8: purine synthesis
2222
Step 9: purine synthesisStep 9: purine synthesis
2323
Step 10: purine synthesis
2424
Step 11Step 11
cyclization or ring closure to form IMPcyclization or ring closure to form IMPwater is eliminatedwater is eliminatedin contrast to step 6 (closure of the in contrast to step 6 (closure of the imidazole ring), this reaction does not imidazole ring), this reaction does not require ATP hydrolysisrequire ATP hydrolysisonce formed, IMP is rapidly converted to once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in AMP and GMP (it does not accumulate in cellscells
2525
Step 11: purine Step 11: purine synthesissynthesis
2626
2727
HN
N N
N
O
Ribose-P
N
N N
N
NH
Ribose-P
COO--OOC
HN
N N
N
O
Ribose-P
O
H
Adenylosuccinate xanthine monophosphate XMP
IMPIMP dehydrogenase
NAD+
NADH
A.S. synthetase
Aspartate + GTP
GDP
N
N N
N
NH2
Ribose-P
HN
N N
N
O
H2N
Ribose-P
A.S. lyase
fumarate
glutamine + ATP
Glutamate + AMP+ PPi
AMP GMP
Synthesis of adenineand guanine nucleotides
28
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
AMP + ATP 2 ADP
GMP + ATP GDP + ADP
accomplished by separate enzymes
GDP + ATP GTP + ADP
same enzyme acts on all nucleotide di & triphosphatesnucleoside diphosphate kinase is an enzyme which playsa key role in the activation of antiviral nucleosides such as Retrovir/AZT
29
30
Ribose-5-P PRPP
ribose-P-pyrophosphokinase5-P-ribosylamine
amidophosphoribosyltransferase
AMP + GMP
ADP + GDP
activation
3131
Salvage of Purines
32
O
HH
OHOH
CH2
H O
OP
O
OH
OH
H
P
O
O-
O P
O
O-
O-
O
HH
OHOH
CH2
H H
OP
O
OH
OH N N
NN
NH2
adenine
PPi
Adenine phosphoribosyltransferase (APRT)
Salvage is needed to maintain the purine pool Salvage is needed to maintain the purine pool (biosynthesis is not completely adequate, especially in (biosynthesis is not completely adequate, especially in neural tissue)neural tissue)
Hypoxanthine-guanine phosphoribosyltransferase Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)(HGPRT)
Hypoxanthine + PRPP Hypoxanthine + PRPP IMP + Ppi IMP + Ppi
Guanine + PRPPGuanine + PRPP GMP + PpiGMP + Ppi
Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of enzyme leads to overproduction of purines which are of enzyme leads to overproduction of purines which are metabolized to uric acid, which damages cellsmetabolized to uric acid, which damages cells
3333
Salvage of PurinesSalvage of Purines
Salvage of purine bases
3434
Salvage of purines
Lesch-Nyhan syndromeLesch-Nyhan syndrome
there is a defect or lack in the HGPRT enzymethere is a defect or lack in the HGPRT enzymethe rate of purine synthesis is increased about the rate of purine synthesis is increased about 200X200Xuric acid level rises and there is gouturic acid level rises and there is goutin addition there are mental aberrationsin addition there are mental aberrationspatients will self-mutilate by biting lips and patients will self-mutilate by biting lips and fingers offfingers off
3535
Lesch-Nyhan syndromeLesch-Nyhan syndrome
3636
3737
Purine AutismPurine Autism
25% of autistic patients may 25% of autistic patients may overproduce purinesoverproduce purines
To diagnose, must test urine over 24 To diagnose, must test urine over 24 hourshours– Biochemical findings from this test Biochemical findings from this test
disappear in adolescencedisappear in adolescence– Must obtain urine specimen in infancy, Must obtain urine specimen in infancy,
but it’s difficult to do!but it’s difficult to do!
3838
3939
Origin of atoms in pyrimidine ringOrigin of atoms in pyrimidine ring
4040
Step 1: synthesis of carbamoyl Step 1: synthesis of carbamoyl phosphatephosphate
Condensation of glutamine, bicarbonate in the Condensation of glutamine, bicarbonate in the presence of ATPpresence of ATP
Carbamoyl phosphate synthetase exists in 2 Carbamoyl phosphate synthetase exists in 2 types: CPS-I which is a mitochondrial enzyme types: CPS-I which is a mitochondrial enzyme and is dedicated to the urea cycle and arginine and is dedicated to the urea cycle and arginine biosynthesis) and CPS-II, a cytosolic enzyme biosynthesis) and CPS-II, a cytosolic enzyme used hereused here
4242
Step 1: pyrimidine synthesisStep 1: pyrimidine synthesis
CPS-II is the major site of regulation in animals: UDP andUTP inhibit the enzyme and ATP and PRPP activate itIt is the committed step in animals
4343
Step 2: synthesis of carbamoyl Step 2: synthesis of carbamoyl aspartateaspartate
enzyme is aspartate transcarbamoylase (ATCase)enzyme is aspartate transcarbamoylase (ATCase)catalyzes the condensation of carbamoyl phosphate catalyzes the condensation of carbamoyl phosphate with aspartate with the release of Piwith aspartate with the release of PiATCase is the major site of regulation in bacteria; it ATCase is the major site of regulation in bacteria; it is activated by ATP and inhibited by CTPis activated by ATP and inhibited by CTPcarbamoyl phosphate is an “activated” compound, so carbamoyl phosphate is an “activated” compound, so no energy input is needed at this stepno energy input is needed at this step
4444
Step 2: pyrimidine synthesisStep 2: pyrimidine synthesis
4545
Step 3: ring closure to form Step 3: ring closure to form dihydroorotatedihydroorotate
enzyme: dihydroorotaseenzyme: dihydroorotase
forms a pyrimidine from carbamoyl forms a pyrimidine from carbamoyl aspartateaspartate
water is released in this processwater is released in this process
4646
Step 3: pyrimidine Step 3: pyrimidine synthesissynthesis
4747
Step 4: oxidation of dihydroorotate Step 4: oxidation of dihydroorotate to orotateto orotate
an irreversible reactionan irreversible reaction
enzyme: dihydroorotate dehydrogenaseenzyme: dihydroorotate dehydrogenase
oxidizing power is derived from quinones oxidizing power is derived from quinones (thru coenzyme Q)(thru coenzyme Q)
4848
Step 4: pyrimidine synthesisStep 4: pyrimidine synthesis
4949
Step 5: pyrimidine synthesisStep 5: pyrimidine synthesis
5050
Step 6: pyrimidine Step 6: pyrimidine synthesissynthesis
5151
The big picture again
5252
Orotic aciduriaOrotic aciduria
an inherited human disease caused by a an inherited human disease caused by a deficiency in the multifunctional enzyme that deficiency in the multifunctional enzyme that catalyzes the last 2 steps in the pyrimidine catalyzes the last 2 steps in the pyrimidine synthesissynthesislarge amounts of orotic acid in urinelarge amounts of orotic acid in urineretarded growth and severe anemiaretarded growth and severe anemiatreat by administration (injection) of uridine treat by administration (injection) of uridine and/or cytidineand/or cytidine
5353
Leflunomide (Arava)Leflunomide (Arava)
Leflunomide is an isoxazole immunomodulatory Leflunomide is an isoxazole immunomodulatory agent agent which inhibits dihydroorotate dehydrogenasewhich inhibits dihydroorotate dehydrogenase)) and has antiproliferative activity. Several and has antiproliferative activity. Several in vivoin vivo and and in vitroin vitro experimental models have experimental models have demonstrated an anti-inflammatory effect. demonstrated an anti-inflammatory effect.
5454
Leflunomide (Arava)Leflunomide (Arava)
5555
Synthesis of uridine and cytidine triphosphate
56
UMP + ATP UDP + ADP
UDP + ATP UTP + ADP
nucleoside diphosphate kinase
CTP synthase (cytidylate synthetase)
N
N
O
H
O
Ribose 3 phosphate
N
NO
Ribose 3 phosphate
NH2glutamine +ATP
Glutamate +ADP +Pi
CTPUTP
(in bacteria, ammonia donates the amino group)
Regulation of pyrimidine nucleotide biosynthesis
57
Glutamine +HCO3
- +ATP
Carbamoylphosphate
carbamoyl phosph.
synthetaseOrotate
OMPUMPUTP + CTP
orotatephosphoribosyltransferase
UTP and CTP are feedback inhibitors of CPS II
Formation of deoxyribonucleotides
58
O
HH
OHOH
CH2
H H
OP
O
OH
OH Base
O
HH
HOH
CH2
H H
OP
O
OH
OH Base
ribonucleotide reductase
dADP, dGDP, dUDP and dCDP are all synthesized by the same enzymeSynthesized from nucleoside diphosphate (not mono or triphosphate) byribonucleotide reductase
Synthesis of dTMPSynthesis of dTMP
Methylation of d-UMP via NMethylation of d-UMP via N55,N,N1010-methylene -methylene THFTHF
Reaction inhibited by 5-fluorouracil Reaction inhibited by 5-fluorouracil (Efudex)(Efudex)
5959
OCH2
H
OH
H
H
H
OPHO
O
OH
N
HN
O
O
F
H
H
HN
N
O
O
F
H
H
5-fluorouracil
Activation of 5-fluorouracil
60
dUMP dTMP
NADPH + H+
NADP+
SERINE
GLYCINE
Regeneration of N5,N10 Methylen THF
DHFN5,N10 – METHYLENE-THFN5,N10 – METHYLENE-THF
THF
dihydrofolate reductaseserine hydroxymethyl transferase
thymidylate synthase
Inhibitors of N5,N10 Methylene THF Regeneration
DHF
THF
FdUMP
dUMP dTMP
NADPH + H+
NADP+SERINE
GLYCINE
N5,N10 – METHYLENE-THF
dihydrofolate reductaseserine hydroxymethyl transferase
METHOTREXATE AMINOPTERIN TRIMETHOPRIM
thymidylate synthase
Hydroxyurea (Hydrea)Hydroxyurea (Hydrea)inhibits the enzyme ribonucleotide reductaseinhibits the enzyme ribonucleotide reductase– DNA synthesis cannot occurDNA synthesis cannot occur– Cell are killed in the S phaseCell are killed in the S phase– Drug holds other cells in the GDrug holds other cells in the G11 phase phase
Primarily used to treat chronic myelogenous Primarily used to treat chronic myelogenous leukemialeukemiaCancer cell develop resistance by:Cancer cell develop resistance by:– increasing quantity of inhibited enzymeincreasing quantity of inhibited enzyme– decreasing sensitivity of enzyme for inhibitordecreasing sensitivity of enzyme for inhibitor
used orallyused orallymajor side effect is leukopeniamajor side effect is leukopenia
6363
H2N
N OH
O
H
HYDROXYUREA
64
N
N
NH2
O
OHOCH2
OH F
F
2',2'-DIFLUORODEOXYCYTIDINE
Another inhibitor of ribonucleotide reductase:indicated for non-small cell lung cancer (usually with cisplatin) also first line treatment for non-resectable pancreatic cancer
65
6666
67
Digestion: RNA + DNA Nucleotides Nucleosides
Base + 1-P-ribosenucleosidase
base + ribose
N
N N
N
Ribose-P
NH2
N
N N
N
Ribose-P
OH
H2O NH3
AMP deaminase
N
N N
N
Ribose
NH2
Nucleotidase
H2O
Pi
HN
N N
N
Ribose
O
Adenosine deaminase
H2O NH3
Nucleotidase
H2O
Pi
Purine nucleosidephosphorylase
HN
N N
N
H
O
may be reusedthroughsalvage pathway
Pi
Ribose-1-P
hypoxanthine
Degradationof AMP
68
PENTOSTATIN
69
N
HOH
N
NN
OHOCH2
OH H
H
H
H
H
H
2'-DEOXYCOFORMYCIN
previously called deoxycoformycin (DCF)
a purine analog with a 7-membered-ring
potent inhibitor of adenosine deaminaseADA is a key enzyme which regulatesadenosine levels in cells
indicated for refractory hairy cell leukemia
other uses: chronic lymphocytic leukemiaand lymphomas
ADA deficiencyADA deficiency
In the absence of ADA lymphocytes are destroyedIn the absence of ADA lymphocytes are destroyeddeoxyadenosine is not destroyed, is converted to deoxyadenosine is not destroyed, is converted to dAMP and then into dATPdAMP and then into dATPdATP is a potent feedback inhibitor of dATP is a potent feedback inhibitor of deoxynucleotide biosynthesisdeoxynucleotide biosynthesisthis leads to SCID (severe combined this leads to SCID (severe combined immunodeficiency disease)immunodeficiency disease)Infants with this deficiency have a high fatality rate Infants with this deficiency have a high fatality rate due to infectionsdue to infections
7070
ADA deficiencyADA deficiency
treatment consists of administering treatment consists of administering pegylated ADA which can remain in the pegylated ADA which can remain in the blood for 1 – 2 weeksblood for 1 – 2 weeks
more efficient is gene therapy: replacing the more efficient is gene therapy: replacing the gene that is missing or defectivegene that is missing or defective
gene therapy has been performed on gene therapy has been performed on selected patientsselected patients
7171
N
N N
N
O
H
Ribose-P
H2N
N
N N
N
O
H
Ribose
H2N
H2O
Pi
N
N N
N
O
H
H
H2N
PNPPi
Ribose-1P
N
N N
N
O
H
Ribose-P
O
H
H2O
Pi
N
N N
N
O
H
Ribose
O
HPi
Ribose-1P
N
N N
N
O
H
H
O
H
H2O NH3
PNP
nucleotidase nucleotidase
Degradation ofGMP and XMP
72
N
N N
N
NH2
H
N
N N
N
OH
H
H2N
N
N N
N
OH
H
N
N N
N
OH
H
HO
ADENINE GUANINE(6-AMINOPURINE) 2-AMINO-6-OXYPURINE)
HYPOXANTHINE(6-OXYPURINE)
XANTHINE(2,6-DIOXYPURINE)
N
N N
N
H
PURINE
N
N N
N
OH
H
HO
OH
URIC ACID(LACTIM FORM)
73
ADENINE + H2O HYPOXANTHINE + AMMONIA
GUANINE + H2O XANTHINE + AMMONIA
HYPOXANTHINE + O2 + H2O
adenase
guanase
XANTHINE + H2O2
XANTHINE + O2 + H20 URIC ACID + H2O2
xanthineoxidase
xanthineoxidase
CATABOLISM OF PURINES
74
N
N N
N
O
H
H
O2 H202
N
N N
N
O
H
H
O
HHYPOXANTHINE
XANTHINE
N
N N
N
O
H
H
O
H
O
H
N
N N
N
O
H
H
O
H
OH
acidic proton
URIC ACID
O2
H202
75
GOUTGOUT
Associated with abnormal amounts of urates in Associated with abnormal amounts of urates in the bodythe bodyEarly stage: recurring acute non- articular Early stage: recurring acute non- articular arthritisarthritisLate stage: chronic deforming polyarthritis and Late stage: chronic deforming polyarthritis and eventual renal complicationeventual renal complicationDisease with rich history dating back to Disease with rich history dating back to ancient Greeceancient Greece
7676
GOUTGOUT
Once fashionable to associate gout with Once fashionable to associate gout with intelligenceintelligence
People with gout:People with gout:– Isaac NewtonIsaac Newton– Benjamin FrankinBenjamin Frankin– Martin LutherMartin Luther– Charles DarwinCharles Darwin
7777
GoutGout
prevail mainly in adult malesprevail mainly in adult malesrarely encountered in premenopausal womenrarely encountered in premenopausal womensymptoms are cause by deposition of crystals symptoms are cause by deposition of crystals of monosodium urate monohydrate (can be of monosodium urate monohydrate (can be seen under polarized light)seen under polarized light)usually affect joints in the lower extremities usually affect joints in the lower extremities (the big toe is the classic site)(the big toe is the classic site)
7878
GoutGout
7979
Diagnostic featuresDiagnostic features
usually affect joints in the lower extremities usually affect joints in the lower extremities ( 95%)( 95%)
onset is fast and suddenonset is fast and sudden
pain is usually severe; joint may be swollen, pain is usually severe; joint may be swollen, red and hotred and hot
attack may be accompanied by fever, attack may be accompanied by fever, leukocytosis and an elevated ESRleukocytosis and an elevated ESR
8080
Drugs which may induce Drugs which may induce hyperuricemiahyperuricemia
niacinniacin
thiazides and other diureticsthiazides and other diuretics
low dose aspirinlow dose aspirin
pyrazinamidepyrazinamide
ethambutolethambutol
cyclosporinecyclosporine
cytotoxic drugscytotoxic drugs
8181
Non-pharmacological Non-pharmacological approachesapproaches
Avoid purine rich foods:Avoid purine rich foods:– red meat and organ meat (liver, kidneys)red meat and organ meat (liver, kidneys)– shellfish, anchovies, mackerel, herringshellfish, anchovies, mackerel, herring– meat extracts and graviesmeat extracts and gravies– peas and beans, aspargus, lentilspeas and beans, aspargus, lentils– beer, lager, other alcoholic beveragesbeer, lager, other alcoholic beverages
Weight lossWeight lossControl alcohol (binge drinking)Control alcohol (binge drinking)
8282
Pharmacological management of Pharmacological management of goutgout
based on the premise that the based on the premise that the hyperuricemia is due to both hyperuricemia is due to both overproduction and underexcretion of uric overproduction and underexcretion of uric acidacidsymptomatic relief of pain is also achieved symptomatic relief of pain is also achieved with analgesics (i.e. indomethacin)with analgesics (i.e. indomethacin)drugs used:drugs used:– analgesics (NSAIDs)analgesics (NSAIDs)– uricosuric agentsuricosuric agents– xanthine oxidase inhibitorsxanthine oxidase inhibitors
8383
Colchicine
84
OCH3
CH3O
CH3O
OCH3
O
N
C CH3
H
O
COLCHICINE
a non-basic alkaloid from the seeds and corms of Colchicum autumnale (Meadow Safron)
COLCHICINECOLCHICINEused in the symptomatic treatment of acute used in the symptomatic treatment of acute attacks of goutattacks of goutdecreases leukocyte motility, decreases decreases leukocyte motility, decreases phagocytosis and lactic acid productionphagocytosis and lactic acid productionnot used in other forms of arthritisnot used in other forms of arthritisa very potent druga very potent drugcan cause severe GI distress and abdominal can cause severe GI distress and abdominal painpain
8585
Inhibits the tubular reabsorption of uric acid Inhibit the tubular excretion of certain organic acid
via the transporter Also used to enhance plasma concentration of certain
anti-infective (beta lactams)
86
SHO2C N
O
O
C3H7
C3H7
PROBENECID (BENEMID)
ALLOPURINOL (Zyloprim)ALLOPURINOL (Zyloprim)
prevention of attacks of gouty arthitis prevention of attacks of gouty arthitis and nephropathyand nephropathyalso used during chemotherapy of also used during chemotherapy of cancer and to prevent recurrent calcium cancer and to prevent recurrent calcium oxalate calculioxalate calculimetabolized to oxypurinol (also an metabolized to oxypurinol (also an inhibitor of xanthine oxidase)inhibitor of xanthine oxidase)inhibits the metabolism of certain inhibits the metabolism of certain anticancer drugs (6-MP, azathioprine)anticancer drugs (6-MP, azathioprine)
8787
88
N
N N
N
OH
H
ALLOPURINOL (ZYLOPRIM)
An inhibitor of xanthine oxidase; prevents the formation of uric acid fromprecursorial purines
8989
Catabolism of a pyrimidine
9090
9191
9292
9393