Download Why study? Genetic disorders of nucleotide metabolsm cause

Nucleotides-Introduction and Purines
10/15/2008 6:14:00 AM
Why study? Genetic disorders of nucleotide metabolsm cause significant and
serious diseases. Cancer cells show an increased production of nucleotides.
Inhibiting nucleotide synthesis inhibits growth. Using this, we can develop
drugs for cancer, antibiotics. Cancer cells are more sensitive to nucleotide
synthesis inhibitors than normal cells.
Roles of Nucleotides
 Nucleotides are the activated precursers for making DNA and RNA
(the genetic material) This makes them prime targets for arresting
growth of cells.
 Activated intermediates in biosynthetic pathways. ATP, CDPglucose, S-adenosyl methionine
 ATP is used as energy currency
 Adenine is part of many coenzymes
 Metabolic regulation: Cyclic AMP
Structure
 A nucleotide has three parts. It has a nitrogenous base (purine or
pyrimidine), a sugar, and 1, 2 or 3 more phosphates. You must
have all three parts to have a nucleotide.

A nucleoside does not have the phosphates (adenosine, uracil, etc)

Three types of bonds
 The base is attached to the suger by an N
glycosidic bond


The first phosphate is contected to the sugar by a
phosphate monoester (ester is an acid to an
alcohol
The phosphates are bound together by acid
anhydride bonds (this is the high energy bond
that is split to give 7 kcal/mole)
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The Bases
 Two flavors: Pyrimidine (one ring) and purine (two ring)



Heterocyclic (carbon and nitrogen are both involved in the ring
Aromatic and planar
Pyrimidines number in a clockwise fashion starting at the bottom
o 1 Nitrogen attached to the sugar
o One urea unit (NCN) and one CCC unit
o Cytosine (two additions to ring), Thymine (three additions to
ring), Uracil (two additions)
 Purines start on the 6 member ring and number 1-6 in a counter
clockwise fashion, then number the other 3 atoms on the 5 member
ring 7, 8, 9
o Sugar is bound at the 9 nitrogen
o One CCC unit sandwiched by two urea units.
o Adenine (one addtion) and Guanine (two additions)
The sugar

If the sugar is ribose, the nucleoside is called a ribonucleoside

If the sugar is deoxyribose, the nucleoside is a deoxyribonucleoside

Five carbon is outside the ring
DNA and RNA are polymers of nucleosides
 A:T
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 G:C
Nucleotides names
 Usually longer than the bases name itself. Apparently that should
help us remember them.
AdenineAdenosine
CytosineCytodine
ThymineThymidine******
GuanineGuanidine
UracilUridine
******Because Thymidine is almost exclusively found in DNA, it is
understood that Thymidine is deoxythymidine, unless otherwise
stated.
Nucleotide names
 Add Nucleoside ________phosphate (fill in blank with mono, di, tri)
Odd Ball Nucleotides
 Intermediates in some of the pathways
 Hypoxanthine (Purine-one addition) (nucleotide is called inosine
monophosphate because scientists are lazy and wont say
hypoxanthine monophosphate)

Xanthine (Purine-two additions)
Purine Synthesis


Although purines and pyrimidines are often available in the diet, the
body likes to make them de novo (from scratch)
Two sources of ribose-5-phosphate
o Oxidative (glucose-6-phosphate dehydrogeogenase) forward
PPP)
o Non oxidative using G3P and F6P (backwards PPP)
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1. First step is PRPP—puts a pyrophosphate on the 1 carbon. This is
an energy using step that requires ATP.
2. Second step (first committed step)—PRPP Glutamyl
Amidotransferase Glutamine is an amino donor. The step cleaves
the pyrophosphate and replaces it with an Amino. Uses water and
glutamine. Adds the N9
3. Third Step—glycine is added and ATP is hydrolyzed and C4, C5 and
N7
4. Formyl group from THF (Formyl Transferase) N10FormylTetrahydrofolate is used and C8 is added
5. N3 is added. Glutamine is the donor
6. Five member ring closure
7. C6 is added by carboxylation
8. Aspartate is added
9. Fumarate is cleaved leaving N1
10. C2 is added by N10Formyl-Tetrahydrofolate
11. Ring Closure—First purine ring formed is Inosine Monophosphate
The next steps differ in the production of Adenosine Monophosphate
and Guanisine Monophosphate
o AMP SynthesisAspartate serves as an amino donor to make
adenosine monophosphate ( two steps: add the entire
aspartate then cleave it off leaving the amino)
o GMP synthesisIMP Dehydrogenase to make Xanthosine
monopphospate and then glutamine is used as an amino
group donor that replaces the carbonyl added by IMP
dehydrogenase
o Regulation of synthesis
 Negative feed back.


First and second step are inhibited by IMP , AMP and
GMP
 PRPP is not purely for Purine synthesis
IMPwhatever step is regulated by its own end produce
 AMP shuts down AMP synthesis
 GMP shuts down GMP synthesis
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Purine Synthesis Overview
 Purine Ring is built into a preexisting ribose-5-monophosphate
(deoxyribose-5-monophosphate)
 Ring atoms are added one at time with the exception of step 3 with
adds an entire glycine
 Glutamine serves as an amino group donor in steps 2, 5 and 15
 Formyl Tetrahydrofolate serves as carbon donor in steps 4 and 10
 The first nucleotide formed is IMP, which is then converted to AMP
or GMP by branched pathways
Glutamine
Rxn 2 (N9)
Rxn 5 (N3)
Rxn 15 (Guanosine)
Aspartate
Rxn 8 (N1)
Rxn 12 (Adenosine)
N10 Tetrahydrofolate
Rxn 4 (C8)
Rxn 10 (C2)
Glycine
Rxn 3 (C4, C5, N7)
Carbon Dioxide
Rxn 7 (C6)
NADH Produced
Step 14 (Guanosine)
ATP Consumed
Rxn 1-PRPP
Rxn 3
Catabolism of Purines
 Adenosine
1. Demamination of C6 (conversion of NH2carbonyl) releases
ammonium and utilizes water. Enzyme is Adenosine
Deaminiase. Deficiency in Adenosine Deaminase is involved
with SCID
2. Purine Nucleoside phosphorylase. Same type of reaction as

glycogen phosphorylase. Removes Ribose 1 phosphate
3. Xanthine Oxidase makes uric acid
Guanosine
1. Guanosine uses Guanase to make xanthine
2. Xanthine Oxidase makes uric acid.
3. Primates stop here- Uric acid may have some antioxidant
properties
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CLINICAL SIGNIFICANCE
Xanthinuria
 Genetic deficiency of Xanthine Oxidase. Causes increased
secreastion of xanthine and hypoxanthine and a decrease in uric
acid. May cause xanthine lithiasis (kidney stones)
Gout-Hyperuricemia
 Hyperuricemia is the excess of uric acid in the blood or urine
 Can crystallize in the joints. Inflammed joints due to depsosition of
uric acid crystals is called gout
 There are some genetic causes
o PRPP sythetase mutation that is not subject to feed back
regulation. If no feeback you make to many purines which
leads to increased purine degredation
o Glucose-6-phopshatse deficiency, increased glucose 6
phosphate leads to increased ribose 5 phosphate via PPP
o Partial HGPRTase deficiency (purine salvage pathway)
Treatment
o Anti-inflammatory
 Colchecine

Alkaloid that inhibits microtubule assembly in
leukocytes (inflammatory response)
 NSAIDs
o Inhibit Uric Acid Production
 Allopurinol inhibits Xanthine Oxidase. Allopurinol is
converted to alloxanthine that inhibits Xanthine Oxidase
o Uricosuric drugs increase clearance of uric acid. Probenecid
increases renal clearance of uric acid by ihibiting reabsorption


Colchecine

Anti-Inflammatory—
Inhibits microtubule
assembly in Leukocytes

NSAIDs

Anti-Inflammatory

Allopurinol

Inhibits Xanthine
Oxidase

Probenecid
(Uricosuric)

Prevents Uric Acid
Reabsorption in the Kidney
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SCID

A deficiency in adenosine Deaminase can Cause Sever Combined
Immunodeficiency syndrome
 Deficit of both T and B cells in the immune system
 May be due to the build up of dATP which inhibits ribonucleotide
Reductase and therefore inhibits DNA synthesis
 First disease to be successfully treated by gene therapy
Purine Nucleoside Phosphorylase Deficiency
 Deficiency in T-cells, B cells are normal
 The problem MAY be from an accumulation of dGTP which can
inhibit reduction of pyrimidine ribonucleotides by inhibit inhibiting
Ribonucleotide Reductase and therefore DNA synthesis
Antimetabolites
 Block metabolic pathways by inhibiting enzyme activity
 Purine Metabolism blockers
o Glutamine Analogs (e.g. Azaserine)
 Inhibit aminotransferases in purine sythesis
 Inhibit the CTP sythtetase in pyrimidine synthesis
 Inhibit enzymes that use glutamine
o Purine Nucleotide Analogs
 Mercaptopurine ihibits phopsphoryl pyrophosphate
amidotransferase (PPRP Glutamyl Amidotransferase)
 Can be used to treat leukemia
o Antifolates
 Folate analogs
 Inhibits the regeneration of formyl tetrahydrofolate
required for steps 4 and 10 of purine synthesis
 Methotrexate, aminopterin

Uses of Antimetabolites
o Antimetabolites are widely used in chemotherapy
o Some antibiotics are antimetabolites
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Nucleotides-Pyrimidines, Deoxyribonucleic Acid, and
Salvage Pathways
10/15/2008 6:14:00 AM
Pyrimidine Biosynthesis
 Start with simple compounds
o Glutamine (N3)
o CO2 (C2)
o ATP
o Aspartate (N1, C6, C5, C4)
 Glutamine, CO2 and ATP make Carbamoyl Phosphate
 Aspartate is added
 All rings are present after the second step


PRPP is used, but in the middle end area. PRPP donates the ribose
phosphate after ring formation
OMP is the first pyrimidine nucleotide then converted to UMP then
to UDP
o UDP is converted to the other end products. The
interconversions must take place at specific phosphorylation
levels
 UDP is phosphorylated to form UTP
 CTP is converted from UTP by glutamine amino donation
 TMP is converted from dUMP
UDP must undergo a reduction and a
dephosphorylation first (Ribonucleotide Reductase
then hydrolysis).
 Enzyme is Thymidylate Synthase
The three major end products are CTP, UTP and dTMP
Synthesis Pathway
1. Carbamoyl Phosphate Synthetase II
i. CO2 + Glutamine glutamate and carbamoyl phosphate
ii. Consumes ATP

iii. C2 and N3
2. Aspartate Transcarbamoylase
i. All members of ring are now present
ii. N1, C6, C5, and C4
3. Dihydrotase
i. Closes the ring
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CARBAMOYL PHOSPHATE II , ASPARTATE TRANSCARBAMOYLASE ,
AND DIHYDROTASE ARE PART OF THE SAME ENZYME (1, 2 and 3)
o Referred to as C.A.D.
o Use substrate channeling, which helps in regulation and
diffusion of substrates from enzyme to ezyme
4. Dihydroorotate Dehydrogenase
i. Make the first pyrimidine-Orotic Acid
ii. Reduces NAD+ to NADH
5. Orotate Phosphoribosyl Transferase
6. Orotidylic Acid Decarboxylase
i. Makes UMP
OROTATE PHOSPHORIBOSYL DEHYDROGENASE AND OROTIDYLIC
ACID DECARBOXYLASE ARE PART OF THE SAME ENZYME (5 and 6)
o Called UMP Synthase
7. UMP is phosphorylated to make UDP. This is where the chain
branches.
i. UDP is reduced and dephosphorylated (ribonucleotide
Reductase) to make dUMP which is then methylated by
methylene THF to make dTMP (Thymidylate Synthase).
ii. UDP is phosphorylated to make UTP which is then converted to
CTP using glutamine as an amino donor (UTPCTP by CTP
synthetase)
Regulation of Pyrimidine Synthesis
 End Feedback—CTP inhibits Carbamoyl Phosphate Synthetase II
o How does can this only regulate CPII and not CPI? Two ways
 Compartmentalization—CPI is in the mitochondria and
CPII is in the cytosol. There are two distinct pools of
Carbamoyl Phosphate.
Metabolic Channeling—Carbamoyl Phosphate is not
diffusing into cytosol, it is just used right away by
Aspartate Transcarbamoylase and then Dihydrotase
Inhibition of Pyrimidine Synthesis
 Glutamine Analogs (Azaserine)—CTP synthetase is inhibited by
glutamine analogs by competitive inhibition

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
5-fluorouracil inhibits thymidylate synthase. 5-fluorouracil is
converted to 5-fluoro-2-deoxyuridine-5-phosphate (FdUMP) which
blocks thymidylate synthase by binding to it irreversibly
Catabolism of Pyrimidines
 Details of the end of the pathway.
 There are two branches of the pathay that converge
 Three end products
o CO2
o NH3
o -Aminoisobutyrate
Can be used to estimate the rate of DNA degradation
and cell death.
CLINICAL RELEVANCE
Hereditary Orotic Aciduria
 Sxmegaloblastic anemia, growth retardation, excess orotic acid in
urine
 Cause is deficiency in two activities
o Orotate phosphoribosyl transferase
o Orotidine-5-P decarboxylase



Pathophysilogy
o Lack of pyrimidines therefore decreased DNA and RNA
synthesis
o Block in pathway decreases concentration of end feedback
mechanism, therefore increased flux through the pathway
(until block)
Txtreat with Uridine
o Kinase will make UTP which will inhibit the pathway and make
CTP
o Kinase will also make UDP so you can make dTMP
Biosynthesis of Deoxyribonucleotides
 Deoxyribonucleotides are made by reduction of already made
diphosphate ribonucleotides
 Reduction can ONLY OCCUR at the diphosphate level
 Ribonucleotide Reductase is the enzyme
o Uses Thioredoxin to reduce
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o Three type of sites that can bind nucleotides
 Catalytic Site-Substrate are any of the ribonucleotides
at the diphosphate level
 Activity Site—allosteric regulator; primarly regulated by
the adenosine nucleotides (ATP, dATP)
 Specifity Site—allosteric regulator; can regulate activity
toward other sites
 dATP binds to the specificity site and that
decreases specificity toward all nucleotides
 ATP binds to specificity site to increase creation of
deoxypyrimidines
 dGTP inhibits pyrimidine deoxyribonucleic acid
synthesis and upregulates ADP conversion to
dADP
 dTTP is not made by ribonucleotide reductase but
can inhibit it allosterically by binding to the
specificity site
 dCTP does not bind to any allosteric site
Why do high levels of thymidine arrest growth of cells?

Pools of dTTP, dGTP, and dATP are increased
o Reason: dTTP increases action of Ribonucleotide Reductase on
GDP which yield dUTP which increases action of
Ribonucleotide Reductase on ADP to yield more dATP
 Pool of dCTP is decreased 10-fold
o Reason: dTTP inhibits reduction of pyrimidine nucleoside
diphophates by ribonucleotide reductase. dTTP can still be
made by salvage pathways, it just cant be made from dUMP
Regulation of Ribonucleotide Reductase


dATP inhibits at this general activity site
dGTP and dTTP act as inhibitors of pyrimidine reduction at the
specificity site
Synthesis of dTMP
 Not made from Ribonucleotide Reductase
 dTMP is made by methylation of dUMP, using methylene-THF s a
cofactor
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Folic Acid Analogs
 Uses basic structure of analogs, but replaces simple R groups with
aminos (and methyls in some cases)
Folate Cycle
 N5N10 Methylene THFFH2 (thymadylate Synthase)
o FH2 is not functional
 FH2 is reduced with NADPH to make THF which then uses serine
hydroxymethyl transferase and converts serineglycine
o Carbon that is donated comes from SERINE! Woooo
o Methotrexate/aminopterin (chemotherapy drugs) block the
dihydrofolate Reductase reaction, therefore THF cannot be
regenerated
How does everything fit together
 Two different pathways (purine and pyrimidine)
 The diphosphates will be converted to deoxyribonucleoties
o dTTP pathway is long and ridiculous
Salvage pathway
 Recycles bases back to nucleotides
 Two types
o Phosphoribosyl transferase
 Adds phospho-sugar in one step
 No enzyme for cytosine (can make from uracil)
o Nucleoside phosphorylases and nucleoside kinases
 Phosphorylase fuses Thymine and deoxyribose1phosphate
 Kinase phosphoyrlates the nucleoside.
CLINICAL RELEVENCE
Lesch-Nyhan Syndrome




Defect in a phosphoribosyl transferase (HGPRT)
Increased uric acid
Neurological presentation (brain relies on salvage pathways for
nucleotide and is therefore starved for guanine nucleotides
Pathphysiology
o Lack of HGPRT reduces salvage of guanine and hypoxanthine,
thus reducing levels of GMP and IMP. PRPP that is normally
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used for salvage pathway is used for purine syntheis, leading
to incrased degredation and therefore more uric acid
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Nucleotides-Objectives
10/15/2008 6:14:00 AM
1. Identify the structure of the five common nitrogenous bases found in
nucleotides that are incorporated into nucleic acids. Name the
nucleosides and nucleotides derived from these bases.
2. Identify the nitrogenous base, sugar, phosphate, glycosidic bond,
phosphomonoester bond and phosphoanhydride bonds of a nucleoside
triphophate
3. Name the Committed Steps in de novo purine and pyrimidine
biosynthesis.
a. Purine—PRPP Glutamyl Amidotransferase (Step 2)
b. Pyrimidine—CAD; Carbamoyl phosphate synthetase II (Step 1)
4. Compare and contrast the biosynthetic pathways for purines and
pyrimidines
a. Purine is built upon the sugar where as pyrimidine is added to sugar
after is has been made
b. Both us PRPP as sourse of ribose
5. Identify the principal metabolic source of one carbon units that are
metabolized via tetrahydrofolate
a. Serine
6. State the first purine nucleotide that is formed during de novo purine
biosynthesis
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a. Inositol Monophosphate
7. State the first pyrimidine nucleotide that is formed during de novo
pyrimidine biosynthesis
a. Orotodylic Monophosphate
8. Name the two steps that are regulated early in the de novo purine
biosynthetic pathway
a. PRPP Synthetase (slightly)
b. PRPP Glutamyl Amidotransferase (major)
9. Name the enzyme that is regulated early in the de novo pyrimidine
biosynthetic pathway of mammals.
a. Caramoyl phosphate synthetase II
10. State the enzyme of nucleotide metabolism that is inhibited by a given
antimetabolite
a. Glutamine Analogs (Azaserine)—inhibits amidotransferases (rxn 2
and rxn 5 of purine synthesis)
b. Purine Nucleotide Analogs (mercaptopurine)—Feed back regulation
of PRPP Glutamyl Amidotransferase
c. Antifolates (methotrexate, aminopterin)—Inhibit the regeneration of
N10-Formyl-THF for Rxn 4 and rxn 10 of purine synthesis and
regeneration of N5 N10 methylene THF for Thymidylate synthase by
inhibiting Dihydrofolate Reductase (DHFR)
d. 5-fluorouracil inhibits Thymidylate Synthetase
11. State the level of phosphorylation at which ribonucleotides are
converted to deoxyribonucleotides
a. Diphosphate level
12. State which nucleotides allosterically inhibit Ribonucleotide Reductase,
and why high levels of thymidine arrest growth of cells in culture
13. Describe the thymidylate synthase reaction and its role in nucleotide
biosynthesis.
a. Thymidylate synthase reaction
14. Diagram the two preformed or salvage pathways of nucleotide
biosynthesis.
a. One step
i. Adds phophosugar to base
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b. Two step
i. Adds base to sugar
ii. Phophorylates sugar
15. State the end product of purine catabolism in man and the precursers
that Xanthine Oxidase acts upon to yield this end product
a. Uric Acid
b. Hypoxanthine and Xanthine
16. State the known enzymatic defects in patients with (and treatments
for these diseases (when available) and the mechanism of action of these
diseases)
a. Gout
i. Causes
1. PRPP Synthetase-mutant that’s not regulated will
increase nucleotide production and consequently
nucleotide degredation
2. Glucose-6-Phosphate Dehydrogenase (leads to
increased ribose-5-phosphate, therefore increased
nucleotides and furthermore increased nucleotide
metabolism)
3. Partial HGPRT deficiency
ii. Treatments
1. Anti-inflammatories (NSAIDS-cyclooxygenase,
Colchecine-microtuble formation in leukocytes)
2. Allopurinol-Inhibits Xanthine Oxidase
3. Uricosuric (probenecid)-inhibits uric acid reabsorption in
the kidney
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b. Lesch-Nyhan Syndrome
i. Defect in HGPRT
c. Orotic Aciduria
i.
d. Xanthinuria
i. Xanthine Oxidase deficiency causes Hypoxanthine and Xanthine
to be excreted in the urine. Can cause Xanthine Lithiasis in
extreme cases.
e. Severe Combined Immunodeficiency Syndrome (SCID)
i. Cause
1. Adenosine Deaminase deficiency-Build up of dATP which
inhibits Ribonucleotide Reductase (makes
deoxyribonucleotides from ribonucleotides), thereby
inhibiting DNA synthesis.
ii. Treatment
1. Gene Therapy-Deliver the gene to the affected tissues
and it may be incorporated, thereby fixing the
deficiency
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