Download Nucleotides: Synthesis and Degredation

Nucleotides: Synthesis and
Degradation
Javad Zavar Reza
Ph.D in Clinical Biochemistry
Department of Biochemistry
School of Medicine
1
Nitrogenous Bases
Planar, aromatic, and heterocyclic
Derived from purine or pyrimidine
Numbering of bases is “unprimed”
2
Nucleic Acid Bases
Purines
Pyrimidines
3
Sugars
Pentoses (5-C sugars)
Numbering of sugars is “primed”
4
Sugars
D-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group
5
Nucleosides
6
Phosphate Groups
Mono-, di- or triphosphates
Phosphates can be bonded to either C3 or C5
atoms of the sugar
7
Nucleotides
Result from linking one or more phosphates with a
nucleoside onto the 5’ end of the molecule through
esterification
8
Nucleotides
Monomers for nucleic acid polymers
Nucleoside Triphosphates are important
energy carriers (ATP, GTP)
Important components of coenzymes
– FAD, NAD+ and Coenzyme A
9
Naming Conventions
Nucleosides:
– Purine nucleosides end in “-sine”
Adenosine, Guanosine
– Pyrimidine nucleosides end in “-dine”
Thymidine, Cytidine, Uridine
Nucleotides:
– Start with the nucleoside name from above and add
“mono-”, “di-”, or “triphosphate”
Adenosine Monophosphate, Cytidine Triphosphate,
Deoxythymidine Diphosphate
10
Nucleotide Biosynthesis
De novo Biosynthesis
Salvage Biosynthesis
11
Nucleotide Metabolism
PURINE RIBONUCLEOTIDES: De novo
– i.e., purines are not initially synthesized as free bases
– First purine derivative formed is Inosine Mono-phosphate
(IMP)
12
13
Steps 1
Step 1:Activation of ribose-5-phosphate
– product: 5-phosphoribosyl-a-pyrophosphate
(PRPP)
– PRPP is also a precursor in the biosynthesis of:
pyrimidine nucleotides
Histidine
Tryptophan
14
Step 1: purine synthesis
15
Step 2: purine synthesis:
commited step
16
Step 3 : purine synthesis
17
Acquisition of purine atom
C8 & purine atom N3
18
Step 6: purine synthesis
19
Step 7: purine synthesis
Acquisition of C6 introduced as HCO3-
20
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-(Nsuccinylocarboxamide)ribotide (SAICAR)
– reaction is driven by hydrolysis of ATP
21
Step 8: purine
synthesis
22
Step 9: purine synthesis
23
Step 10: purine
synthesis
24
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
25
Step 11: purine
synthesis
26
27
O
N
HN
Synthesis of
adenine
and guanine
nucleotides
Aspartate + GTP
N
N
Ribose-P
IMP
GDP
IMP dehydrogenase
NAD+
A.S.
synthetase
NADH
COO-
O
-OOC
NH
N
HN
N
N
O
Ribose-P
H
N
N
N
N
Ribose-P
xanthine monophosphate
XMP
Adenylosuccinate
glutamine + ATP
A.S.
lyase
fumarate
Glutamate + AMP
+ PPi
O
NH2
N
N
N
N
N
HN
H2 N
N
Ribose-P
28
Ribose-P
AMP
N
GMP
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)
AMP + ATP
2 ADP
accomplished by separate enzymes
GMP + ATP
GDP + ADP
- nucleoside diphosphate kinase
GDP + ATP
GTP + ADP
same enzyme acts on all nucleotide di & triphosphates
nucleoside diphosphate kinase is an enzyme which plays
a key role in the activation of antiviral nucleosides such as Retrovir/AZT
29
activation
amidophosphoribosyl
transferase
ribose-P-pyrophosphokinase
Ribose-5-P
5-P-ribosylamine
PRPP
AMP + GMP
ADP + GDP
30
31
Salvage of Purines
OH
OH
P
O
H
CH 2
O
O
H
O
H
O
H
OH
OH
P
O-
O
O
P
adenine
OPPi
O-
NH2
N
Adenine
phosphoribosyltransferase
(APRT)
OH
OH
P
N
O
CH 2
O
O
H
H
H
H
OH
32
OH
N
N
Salvage of Purines
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
33
Salvage of purine bases
Salvage of purines
34
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
35
Lesch-Nyhan syndrome
36
37
Purine Autism
25% of autistic patients may
overproduce purines
To diagnose, must test urine over 24
hours
– Biochemical findings from this test
disappear in adolescence
– Must obtain urine specimen in infancy,
but it’s difficult to do!
38
39
Origin of atoms in pyrimidine ring
40
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
42
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
43
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
44
Step 2: pyrimidine synthesis
45
Step 3: ring closure to form
dihydroorotate
enzyme: dihydroorotase
forms a pyrimidine from carbamoyl
aspartate
water is released in this process
46
Step 3: pyrimidine
synthesis
47
Step 4: oxidation of dihydroorotate
to orotate
an irreversible reaction
enzyme: dihydroorotate dehydrogenase
oxidizing power is derived from quinones
(thru coenzyme Q)
48
Step 4: pyrimidine synthesis
49
Step 5: pyrimidine synthesis
50
Step 6: pyrimidine
synthesis
51
The big picture again
52
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
53
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.
54
Leflunomide (Arava)
55
Synthesis of uridine and
cytidine triphosphate
UMP + ATP
UDP + ADP
nucleoside diphosphate kinase
UTP + ADP
UDP + ATP
CTP synthase (cytidylate synthetase)
O
H
glutamine +
ATP
Glutamate +
ADP +Pi
NH2
N
O
N
N
Ribose 3 phosphate
UTP
(in56 bacteria, ammonia donates the amino group)
O
N
Ribose 3 phosphate
CTP
Regulation of pyrimidine nucleotide
biosynthesis
Glutamine +
HCO 3- +
ATP
carbamoyl phosph.
synthetase
Orotate
orotate
phosphoribosyl
transferase
UTP + CTP
UTP and CTP are feedback inhibitors of CPS II
57
Carbamoyl
phosphate
UMP
OMP
Formation of
deoxyribonucleotides
ribonucleotide reductase
OH
OH
P
Base
O
CH2
O
OH
O
H
H
H
OH
P
Base
O
CH2
O
H
OH
OH
O
H
H
H
H
OH
H
dADP, dGDP, dUDP and dCDP are all synthesized by the same enzyme
Synthesized from nucleoside diphosphate (not mono or triphosphate) by
ribonucleotide reductase
58
Synthesis of dTMP
Methylation of d-UMP via N5,N10-methylene
THF
Reaction inhibited by 5-fluorouracil
(Efudex)
59
Activation of 5-fluorouracil
O
F
HN
O
O
HO
P
O
O
CH2
O
F
HN
OH
H
O
N
N
H
H
H
H
H
OH
5-fluorouracil
60
H
H
Regeneration of N5,N10 Methylen THF
dUMP
dTMP
thymidylate synthase
DHF
N5,N10 – METHYLENE-THF
NADPH + H+
GLYCINE
dihydrofolate reductase
serine hydroxymethyl
transferase
NADP+
SERINE
THF
Inhibitors of N5,N10 Methylene THF Regeneration
METHOTREXATE
AMINOPTERIN
TRIMETHOPRIM
dUMP
dTMP
DHF
thymidylate synthase
N5,N10 – METHYLENE-THF
FdUMP
NADPH + H+
GLYCINE
serine hydroxymethyl
transferase
SERINE
THF
dihydrofolate reductase
NADP+
Hydroxyurea (Hydrea)
inhibits the enzyme ribonucleotide reductase
– 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
H
N
OH
H2 N
O
HYDROXYUREA
63
NH2
N
O
HOCH 2
N
O
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
64
65
66
Digestion: RNA + DNA
Nucleosides
Nucleotides
nucleosidase
Base + 1-P-ribose
base + ribose
67
Degradation
of AMP
OH
NH2
NH3
H2 O
N
N
N
N
AMP deaminase
N
N
N
N
Ribose-P
Ribose-P
H2 O
H2O
Nucleotidase
Nucleotidase
Pi
Pi
O
NH2
NH3
H2 O
N
N
HN
N
Adenosine deaminase
N
N
N
Pi
N
Ribose
Ribose-1-P
Ribose
Purine nucleoside
phosphorylase
O
may be reused
through
salvage pathway
N
HN
N
N
H
hypoxanthine
68
PENTOSTATIN
previously called deoxycoformycin (DCF)
a purine analog with a 7-membered-ring
H
OH
H
N
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
69
N
N
N
HOCH2
O
H
H
H
H
OH
H
2'-DEOXYCOFORMYCIN
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
70
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
71
O
O
H
N
N
H2N
N
H
N
O
N
N
Ribose-P
N
N
H
Ribose-P
H2O
H2O
nucleotidase
nucleotidase
Pi
Degradation of
GMP and XMP
Pi
O
O
H
N
N
N
N
N
N
H2N
H
O
Pi
N
N
Ribose
PNP
Ribose
H
Ribose-1P
Pi
PNP
Ribose-1P
O
O
H
N
N
H
N
N
H2N
72
N
N
H
H2O
NH3
O
N
N
H
H
NH2
OH
N
N
N
N
N
N
N
H
GUANINE
2-AMINO-6-OXYPURINE)
ADENINE
(6-AMINOPURINE)
N
PURINE
OH
N
N
N
H
OH
OH
N
N
N
H2N
H
N
N
N
N
OH
N
N
H
HYPOXANTHINE
(6-OXYPURINE)
73
HO
N
N
H
XANTHINE
(2,6-DIOXYPURINE)
HO
N
N
H
URIC ACID
(LACTIM FORM)
CATABOLISM OF PURINES
adenase
ADENINE + H 2O
GUANINE + H 2O
HYPOXANTHINE + AMMONIA
guanase
HYPOXANTHINE + O
XANTHINE + O
74
2
2 + H 2O
+ H 20
XANTHINE + AMMONIA
xanthine
oxidase
xanthine
oxidase
XANTHINE + H 2O 2
URIC ACID + H 2O 2
O
O
H
H
N
N
N
N
N
N
O2
H
O
H202
N
N
H
H
HYPOXANTHINE
XANTHINE
O2
H202
O
H
O
N
N
H
O
N
H
75
N
N
OH
H
O
N
O
N
N
H
acidic proton
H
URIC ACID
H
GOUT
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
76
GOUT
Once fashionable to associate gout with
intelligence
People with gout:
–
–
–
–
Isaac Newton
Benjamin Frankin
Martin Luther
Charles Darwin
77
Gout
prevail 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)
78
Gout
79
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
80
Drugs which may induce
hyperuricemia
niacin
thiazides and other diuretics
low dose aspirin
pyrazinamide
ethambutol
cyclosporine
cytotoxic drugs
81
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)
82
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
83
Colchicine
CH 3O
H
N
C
CH 3O
OCH 3
CH 3
O
O
OCH 3
COLCHICINE
a non-basic alkaloid from the seeds and corms of Colchicum autumnale
(Meadow Safron)
84
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
85
O
HO2C
S
O
C3H7
N
C3H7
PROBENECID (BENEMID)
 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
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)
87
OH
N
N
N
N
H
ALLOPURINOL (ZYLOPRIM)
An inhibitor of xanthine oxidase; prevents the formation of uric acid from
precursorial purines
88
89
Catabolism of a pyrimidine
90
91
92
93