Finish Nucleotide Metabolism!

Last Time:
Purine synthesis
Pyrimidine synthesis
dNDP synthesis

This Time:
dTTP
Salvage Pathways
Nucleotide metabolism and
disease
Let’s have one more
Lecture on
metabolism and
then we’ll go
Last Time:
Synthesis of Purines and Pyrimidines
The Road to AMP and GMP is branched at IMP
The Road to CTP goes through UMP
Feedback inhibition - regulates levels of NTP concentrations

HCO3-
Gly
Asp Pyrimidine Ring is
N10-Formyl
N10-Formyl
built first - then
THF
THF attached to ribose

Amide N
of Gln Amide N
of Gln
Purines are built
Asp
stepwise on a HCO3-
ribose-5’-P platform

2
Last Time:
Ribonucleotide Reductase keeps dNDP synthesis in balance
specificity what binds here controls the type of
catalytic
rNDP bound at the catalytic site
site site

activity
Sequence of NDP
α-subunits site
conversion:
1) UDP/CDP, 

what binds here 2)GDP, 

controls the overall & 3) ADP
activity of the enzyme -
the on/off switch When dATP levels are
ATP -on / dATP-off high enough the
α-subunit enzyme is switched
β-subunits off
One Catalytic site &
Two allosteric sites
β-subunits
RNR subunits can exist in three different states
Generate a Tyr radical 1. free α2- and β2-dimers (inactive)
Transfers to the α-active 2. active α2β2 tetramer
site over 35 Å away 3. inactive α4β4 octamer 3
Biosynthesis of dTTP from UMP: need 5 enzymes
(dTTP specific for DNA synthesis)
O
1. ribonucleotide kinase (UMP kinase) O
H3 C
2. ribonucleotide reductase (RNR)
NH
NH

N O
3. nucleotide diphosphate kinase (NDP Kinase)
O O N O
O O
O O
P P
O O
O
HO OH
4. dUTP phosphatase (dUTPase) O
HO

5. thymidylate synthase
1 2 3 4
UMP UDP dUDP dUTP dUMP

Why dUMP? Why not go straight from dUTP to dTTP?

dUTPase is a very active diphosphorylase

4
dUTP dUMP + PPi

Keeps dUTP concentration low
Prevents incorporation of dUTP into DNA by DNA polymerase
Energy is used to prevent errors!
4
Step 5 in the Biosynthesis of dTTP from UMP
(oxidation of THF accompanies carbon transfer)
Tetrahydrofolate
Serine (THF)
NADP+

Glycine Dihydrofolate
Reductase
N5,N10-
methylene THF NADPH

Dihydrofolate

Thymidylate
Kinase

dTDP
NDK
dUTP dUMP dTMP
Thymidylate
dTTP
dCMP Synthase
NH4+
H2O 5
Nucleoside Bases are degraded to Uric Acid
or β-Alanine and NH4+

Nucleic Acids
Endonucleases
P
P P
Oligonucleotides
P
Exonucleases
Nucleoside-5’-
Pi monophosphates

Nucleosides Ribose-1’-P O

H3N O
Nucleoside
phosphorylase
Nucleobases
Pi Purine Pyrimidine NH4+, CO2
bases bases

Degradation
β-Ureido-
Uric Acid
proprionic acid
6
Nucleotides & Nucleobases can be Recycled via Salvage Pathways
Sources: Dietary, Nucleic Acid Turnover, DNA damage

P P P
P P
P
Nucleic Acids P P P
P P
P
P
P P
Nucleoside
Kinases triphosphates
P (base specific
Nucleoside-5’- kinases and NDK)
Salvage
monophosphates
PP
Phosphoribosyl
transferases
Nucleobases PRPP
Purine Pyrimidine NH2
O

bases bases N
HN
O
N O
H O N
N H
NH

HN N NH2 NH2

Degradation N
N

HN N

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Degradation of purines produces Uric Acid

Guanine

NH3
Adenosine

Hypo-
Inosine Xanthine Uric Acid
Xanthine
Xanthine
Adenosine must be deaminated
oxidase
to Inosine before ribose-1-P can
FAD, Mo, 

be removed by nucleoside
Fe-S Clusters
phosphorylase

High circulating levels of Uric Acid can lead to Gout
• Urate (uric acid) is not very soluble
• Precipitation of Sodium Urate crystals in joints and kidneys
• Painful condition – 1% of the population – mostly males
• Inhibitors of Xanthine Oxidase can be used to treat Gout
8
Treatment of excess uric acid production
Xanthine oxidase (XO) inhibitors
Substrate Inhibitor

Hypoxanthine Allopurinol
Xanthine
oxidase
FAD, Mo, 

Xanthine Fe-S Clusters
Alloxanthine

Alloxanthine binds tightly to Xanthine Oxidase
• Suicide (mechanism-based) inhibitor of uric acid
synthesis
• Increases levels of precursors that are more
soluble
Non-Purine inhibitor (Uloric)
• Introduced 2009
• STRONGLY binds to the Mo center of XO
• Effective at lower concentrations
9
Defects in Purine Salvage Pathways can have severe metabolic
consequences
> 90% of Purine bases are recycled

P P P
P P
Nucleic Acids P
P P P
P P
P
P
P P Nucleoside
Oligonucleotides
P Kinases triphosphates
Nucleoside-5’-
monophosphates Salvage
PP
Nucleosides Ribose-1-P Phosphoribosyl
transferases
Adenosine Nucleobases PRPP
Deaminase Purine Pyrimidine
Nucleoside bases bases
phosphorylase
XO

Degradation
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Lesch-Nyhan Syndrome – Deficiency in Purine Salvage

Adenine Hypoxanthine Xanthine Guanine

Uric Acid

PRPP PRPP

Purine salvage Hypoxanthine-Guanine
Adenine Phospho- Phospho-Ribosyl
Ribosyl Transferase Pathways Transferase (HGPRT)
(APRT)

AMP ATP ATP or GTP IMP or GMP

Complete loss of HGPRT activity is severe (X-linked recessive)
• Guanine and Hypoxanthine are consistently produced due to turnover of nucleic acids
• No salvage of guanine or hypoxanthine – Increased de novo synthesis of purines
• Excess production of uric acid – severe gouty arthritis
• Nervous system disorders - Motor disabilities, learning disabilities and behavioral
disorders
• Severe aggression and self-mutilation
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Adenosine Deaminase - degradation of purines to uric acid
(works on both Adenosine and deoxy-Adenosine)
Adenosine

Inosine HypoXanthine XO Uric Acid

Adenosine
deoxy-Adenosine

Inactive Adenosine Deaminase
causes a metabolic domino
Adenosine effect
Deaminase
• Accumulate Adenosine and
deoxyAdenosine
• Salvaged to ATP and dATP
• ↑ [dATP] turns RNR off
• dNTP pools knocked out of
balance
Inosine deoxy-Inosine • Cells can’t grow and divide
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Adenosine Deaminase (ADA) deficiency:
Impaired Purine Degradation and Salvage
Severe Combined Immunodeficiency Syndrome (SCID)

Known forms of
SCID

X-linked SCID
David Vetter
Jak3
ADA (15%)
IL-7R • B and T Lymphocytes can’t
proliferate
CD3 δ or ε
• Can’t mount an immune
RAG1/RAG2
response
CD45
• Patients are highly susceptible
to infection

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ADA deficiency can be successfully treated with 

Gene-Therapy
Enzyme replacement therapy Bone marrow transplants
Patients are injected twice weekly (Disease affects Lymphocytes)
with ADA Painful & matches are hard to find
Expensive life-long process Common Treatment

Blood (2012) 129: 3635-3646

Gene-replacement therapy
• 11 year clinical trial
• Bone marrow cells were removed and modified with a virus harboring
the intact ADA gene
• Light dose of chemotherapy to remove stem cells that do not produce ADA
• Re-introduce modified bone marrow cells
• Immune systems were restored in 50% of the patients
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Nucleotide metabolism provides targets for clinical
intervention
Chemotherapy – goal is to selectively target a metabolic process
that is specific to the disease.

Rapidly proliferating cells
susceptible to disruptions in nucleotide metabolism
require nucleotides for growth and DNA synthesis

Selectively target Cancer cells, bacterial and viral infections

In mature adults, most cells are not dividing
will not be affected as much

Notable exceptions –
bone marrow cells (blood and immune cells), hair cells,
intestinal mucosa

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Inhibitors of Glutamine Amidotransferases are potent drugs

These compounds interfere
Glutamine with reactions that require
Gln as a nitrogen donor
(Gln amidotransferases)

Azaserine At least half a dozen reactions
require Gln during the
synthesis of purines and
pyrimidines
Acivicin
Azaserine has antibiotic and
anti-tumor properties
NH3 released
from Gln Acivicin is an anti-tumor
travels via a agent
channel to a
second active
site
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Thymidylate Synthase is a target for Chemotherapy
N5,N10-methylene THF

Dihydrofolate

dTDP

dUMP dTMP
Thymidylate
dTTP
Synthase

• Inhibit Thymidylate Synthase - Block production of dTTP
• Lethal to rapidly dividing cancer cells that need to
synthesize DNA
• 5-fluorouracil and 5-fluorodeoxyuridine are inhibitors of
Thymidylate Synthase
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Thymidylate Synthase forms a covalent substrate-cofactor H2N
NH

intermediate during catalysis DHF
N NH

H2 N H2 N O N C
N5,N10- NH NH
NH NH
THF N N HN

H R
N C N C H H2 N
O O
H NH H
H C O C
N HN N N
H H
O R HN R
H H
H :B O N C

dUMP HN 6
:B
O N
H
SS O H
HN
5 O O
O N C H
H
O Cys HN R
O
O :S P O
H:B
O
Cys HO O N
O P O
O
H S
S

O O
HO Cys
Enzyme/Substrate/Cofactor O P O

Covalent Complex
O HO

• The C-5 of dUMP is made nucleophilic by thiolate addition at C-6
(An active site cysteine forms a covalent intermediate with dUMP at C-6)
• C-5 can attack the N5,N10 methylene-THF forming a carbon-carbon bond

(dUMP, THF, and active site Cys are now covalently linked)
• Removal of the C-5 proton breaks the Carbon-Nitrogen bond to the cofactor
• This is followed by hydride transfer (not shown) to form dTMP and DHF
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Suicide Inhibition of Thymidylate Synthase by
5-fluoro-deoxyuridylate
H2 N
NH
N NH

FUra FdUrd O N C H
H
O C HN
H
salvage HN
FH R

pathway :B
O N
H SS
O O

O P
Cys
O
O HO

Irreversible mechanism
5-fluorodeoxyuridine 5’- based inhibitor
monophosphate (FdUMP) (TS can’t abstract F+ ion)

5-fluoro-uracil (FUra) & 5-fluorouridine (FdUrd) are “pro-drugs”
Not 100% selective
5-fluoro-uracil can be salvaged and incorporated into ribonucleotides
and RNA in non-proliferating cells 19
Inhibition of Thymidylate Synthase by anti-Folates
(analogs of THF)
Raltitrexed
dUMP

Pub ID: 1HVY

Raltitrexed competes with N 5,N10-THF for binding to TS (Ki < 9nM)
Used clinically for certain cancers
Most Anti-Folates target TS or DHFR
• Methotrexate - Antineoplastic & immunosuppressant
• Pemetrexed - Anti-neoplastic
• Proguanil - Anti-malarial
• Pyrimethamine - Anti-protozoal
• Trimethoprim - Broad-spectrum anti-microbial
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Inhibition of Dihydrofolate Reductase (DHFR)
Tetrahydrofolate
(THF)
NADP+

Dihydrofolate
N5,10-THF Reductase

NADPH
dUMP TS
Dihydrofolate
dTMP

TS transfers a 1-carbon unit AND OXIDIZES the THF cofactor to DHF
(Only known reaction where this happens)

Dihydrofolate Reductase (DHFR) replenishes THF pools
Inhibit DHFR and deplete THF, stops synthesis of dTTP
Effective target for cancer treatment
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Inhibition of Dihydrofolate Reductase (DHFR)

NADH
Folate

NADH
Methotrexate DHF

Methotrexate is a tight-binding
substrate analog of DHF

1000X greater affinity for DHFR
than the natural substrate DHF

Low doses - rheumatoid arthritis Methotrexate
High doses - cancer treatment
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Inhibition of Bacterial DHFR with Trimethoprim 

(an antibiotic)

Pyrimidine inhibitor of DHFR
(anti-folate)

Binds the same site as
Methotrexate

NADH
Binds as much as 30000X better
to certain bacterial DHFRs than
Trimethoprim
the human enzyme

An effective Antibiotic

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George H. Hitchings – born in Hoquiam Washington
Received a BA and Masters degree in Chemistry from the UW (1927 and 1928)
In 1942 he began work for Wellcome Research Laboratories. Gertrude Elion
joined his team in 1944
In 1988 George Hitchings and Gertrude Elion, were awarded the Nobel Prize in
Medicine for their discoveries of "important principles for drug treatment"
Purine and Pyrimidine Indications
Analog
6-Mercaptopurine, Purine Analog Leukemia
thioguanine
Azathioprine Purine Analog Autoimmune diseases/Organ transplant rejection
Allopurinol Purine Analog (XO) Gout, Uric acid accumulation
Pyrimethamine Inhibits DHFR Malaria
Trimethoprim Inhibits DHFR Bacterial infections (urinary tract)
Pulmonary infections after transplants
Acyclovir Guanosine Analog Herpes Virus / AIDs / Shingles

“While drug development had earlier mainly been built on chemical
modification of natural products they introduced a more rational approach
based on the understanding of basic biochemical and physiological processes.”

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Now you can celebrate!