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Med1C Villiran – Nucleotide Metabolism Biochem 1b

Let’s Talk Nucleotides


Different Roles of Nucleotides
Essential for all cells
Dietarily nonessential - Syn’d from amphibolic intermediates

 Ribonucleoside and deoxyribonucleoside phosphate


o Activated precursors of DNA and RNA
 Serve as carriers of activated intermediates in the synthesis of some carbohydrates, lipids and proteins
o UDP-glucose: active nucleotide for glycogen syn
o CDP-glycerol: active nucleotide substrate for phosphoglceride syn
o S-adenosyl methionine: active methyl donor in methylation rxns
 Adenine nucleotides are structural component of 3 major coenzymes:
o coenzyme A
o FAD
o NAD
o NADP (I guess this one’s minor?)
 Metabolic regulator for many intermediary pathways, inhibiting or activating key enzymes.
o cAMP – IC 2ndary messenger
o cGMP – IC signal or 2ndary messenger
 could act antagonistically to cAMP
 “Energy currency” in the cell
o ATP: universal currency made from oxidation of foodstuffs
o GTP: involved in translocation of nascent peptide chains in ribosomes during Tnl of proteins and activation of
signal coupling proteins

Nucleotide Structure

Numbering
 Carbon and nitrogen atoms in the rings of the base and the sugar are numbered separately
 Atoms in the rings of the bases are numbered
o 1-6 in PYRIMIDINE and 1-9 in PURINE
 Carbons in the pentose are numbered 1-5

Building a Nucleic Acid


 Add’n of pentose sugar to a base  nucleoside
 Mono, di or triphosphate esters of nucleosides
 Phosphate group attached by linkage to the 5’-OH of the pentose  nucleoside 5’-phosphate or 5’nucleotide
 Type of pentose denoted prefix in the names “5’ ribonucleotide and 5’ deoxyribonucleotide”
 Phosphate group are responsible for the negative changes associated with nucleotides and nucleic acids

Lecture, Manual 1 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

Possible base modifications


• Methylation
• Hydroxymethylation
• Glycosylation
• Acetylation
• Alterations of the atoms in pyrimidine ring
• Presence of unusual base in a nucleotide sequence
o aid in its recognition by specific enzyme
o protect it from being degraded by nucleases

Biomedical Importance
 Biosyn regulated and coord’d by feedback mechanisms
 Production varies, maybe in accord w/physio demand
 Crazy image of nucleotide pathways: see manual, pg 101

Fate of Ingested
Nucleic acids and nucleotides
 NOT incorporated directly into tissue nucleic acids
 Degraded in GIT
o First into MONOnucleoTides  by ribonucleases, deoxyribonucleases and polycleotidases
o Second into nucleoSides  by nucleotidases and phosphatases via hydrolysis
o Then, absorbed
or
o Further degradaded into purine and pyrimidine bases  by intestinal phosphorylase
 Purine bases –oxidized uric acid
o Can be absorbed and excreted in urine
 Degradation of dietary nucleic acids in the small intestine (see image pg 116)

Now, Let’s Talk About Purines & Pyrimidines

De Novo Biosyn of Nucleotides

Origin of PURINE atoms


 Amino acids
o N3 and N9 from Gln
o C4, C5, C7 from Gly
o N1 from Asp
 CO2 – C6
 Derivatives of tetrahydrofolate
o C3 from N10-formyl-FH4
o C8 from N5N10-methenyl-FH4

Origin of PYRIMIDINE atoms


 Glutamine – N3
 CO2 – C2
 Aspartic acid – N1, C4, C5, C6
 Other sources also state that N3 and C2 are from Carbamoyl-P
(made by the CO2 and Gln)

Lecture, Manual 2 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

De Novo Biosyn vs. Salvage Pathways

See Img Manual pg 115


Just know that you have the two options, and then of course there is also straight up degradation but that salvage being “cheaper”
energy-wise is preferred and salvage products (nucleic acids) will thus via negative feedback will inhibit de novo syn.

Salvage Pathways
Salvage pathways are important for cells that don’t have PRPP glutamyl amido ransferase – i.e. RBCs

Purines and Pyrimidines from


 Normal turnover of cellular nucleic acids
or
 obtained from diet that are not degraded
 Can be reconverted (SALVAGED) into nucleoside triphosphate (NTP)s
 Energetically much less expensive than complete de novo synthesis
 Pathway for both is essentially the same, but different enzume
o Purines: adenine phosphoribosyl transferase and hypoxanthine-guanine phosphoribosytransferase
o Pyrimidines: pyrimidine phosphoribosyltransferase
 Both utilize PRPP as source of ribose-P

Salvage Pathways of Purine & Pyrimidine Bases (Manual, pg 114)

Free Base (Purine & Pyrmidine)

Ribose-1-PO4

PRPP
Pi

PPi Nucleoside

NMP

Kinase
ATP
NDP
NMP

Kinase
ATP
NMP
NMP NTP

Lecture, Manual 3 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

Ok, Fine. We can just talk about PURINES.

De nove PURINE Nucleotide Biosyn


Step 1: 5-phosphoribosyl-alpha-pyrophosphate (PRPP) synthesis from ribose-5-phosphate and ATP by ribose-5-
phosphate pyrophosphokinase (PRPP synthetase).
 PRPP synthetase
o Activated by Pi
o Inhibited by purine nucleoside di & triphosphates.

Step 2: 5-Phosphoribosyl-b-1-amine synthesis from a-PRPP, glutamine, and H2O by glutamine phosphoribosyl
pyrophosphate amidotransferase.
 The amide of glutamine replaces the pyrophosphate group attached to carbon 1 of PRPP.
 Glutamine phosphoribosyl pyrophosphate amidotransferase
o Inhibited by the
purine 5’-
nucleotides
AMP,GMP and
IMP, the end
products of this
pathway.
o This is the
committed step
in purine
nucleotide
biosynthesis

The whole process creates


Inosine Monophosphate (IMP) –
the “parent” purine nucleotide
 requires 4 ATP
 Inhibitors of PURINE
synthesis
o Specific for
inhibiting the
growth of rapidly
dividing
microorganisms
(ex. Sulfonamides,
folic acid analogs)

Lecture, Manual 4 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

IMP  GMP or AMP


 Two-step, energy requiring pathway.
 Note that the synthesis of AMP requires GTP as an energy source, whereas the synthesis of GMP requires ATP
 The 1st rxn in each pathway is inhibited by the end product of that pathway
o provides a mxn for diverting IMP to the syn of the species of purine present in lesser amounts.

Add’n of more Phosphates


 Nucleoside DIphosphates are syn’d from the corresponding nucleoside MONOphosphates by base-specific nucleoside
monophosphate kinases.
 ATP is used as energy source because >concentration than other nucleoside triphosphates
o For example, adenylate kinase: AMP + ATP ↔ 2 ADP
o For example, guanylate kinase: GMP + ATP ↔GDP + ADP
o Adenylate kinase is particularly active in liver and muscle
 turnover of energy from ATP is high.
 Function: maintain an equilibrium among AMP,ADP, and ATP:
 2ADP ↔ AMP + ATP
 Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase
o unlike monophosphate kinases, had broad specificity.
o For example, GDP + ATP ↔ GTP + ADP
o For example, CDP + ATP ↔ CTP + ADP

Lecture, Manual 5 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

Salvage Pathway of PURINE


Deficiency in HGPRT causes Lesch-Nyhan syndrome – tbd below in Diseases

Enzymes involved

Two key transferase enzymes are involved in the salvage of purines:


 1. Adenosine phosphoribosyltransferase (APRT), which catalyzes the following reaction:
o adenine + PRPP <-----> AMP + PPi
o (no image, use your imagination)
 2. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the following reactions:
o hypoxanthine + PRPP <------> IMP + PPi
o guanine + PRPP <--------> GMP + PPi
 Both utilize PRPP as source of ribose-5-phosphate group
 Release of pyrophosphate makes these reactions irreversible

Lecture, Manual 6 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

Catabolism (Degradation) of Adenine & Guanosine


 Degraded by removal or alterations of portions of the nucleotide
 End product in human: Uric Acid
o Other mammals oxidize uric acid to allantoin which can further be degraded to urea or ammonia
o Formation of Uric Acid

Lecture, Manual 7 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

Different Drugs involved in PURINE Metabolism – Cancer Chemotherapy


Remember: drugs w/c inhibit nucleotide metab inhibit growth of cancer cells but non-specific ones may also impair healthy tissue.

1. 6-Mercaptopurine (6MP)

 Antitumor drug
 Analogue of adenine
 Metabolized to a ribonucleotide by the APRT salvage pathway  inhibiting the conversion of IMP to GMP or AMP
 Also inhibits the rate limiting step of the purine de novo pathway
 Simultaneous administration with allopurinol potentiates its effect as 6-Mercaptopurine will not be degraded and
therefore will delay its inactivation.

2. Adenosine arabinoside (the sugar is replaced by an arabinose)

 Antiviral and an antitumor drug in man.


 Inhibits DNA polymerase after its conversion to the triphosphate form.

3. Azaserine

 Analogue of glutamine
 Inhibits
o incorporation of N3 and N9 into the purine ring (de novo biosynthesis
o formation of GMP from IMP, CTP from UTP-all reactions requiring the entry of glutamine.

Different Diseases of PURINE Metabolism


Gout
 Inability to regulate production of purine nucleotides in any way presents clinically as GOUT but likely to be
o Overactivity of PRPP synthetase or PRPP amido transferase
o Overproduction and overexcretion of purine catabolites or resistance to feedback inhibition.
 e.g. An elevated Vmax increased affinity for ribose 5-phosphate
 Gouty Arthritis : charac’d by deposition of tophi in soft tissues and joins

Lesch-Nyhan syndrome (see img in Purine salvage)

 Hyperuricemia charac’d by frequent episodes of uric acid lithiasis & a bizarre syndrome of self mutilation
 Complete deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
o Inability to salvage hypoxanthine or guanine
 levels of PRPP  purine overproduction  Excessive production of uric acid and IMP and GMP and de
novo syn
 Mutations include deletions, frameshift mutations, base substitutions, and aberrant mRNA splicing.

Von Gierke’s Disease

 SECONDARY, Purine overproduction and hyperuricemia


 Glucose-6-phosphatase deficiency  glucose-6-phosphate  HMP shunt  IC ribose-1-phosphate  cellular
prodxn of PRPP  over produxn of purine nucleotides  when degraded  Hyperuricemia

Hypouricemia

 Hypouricaemia and excretion of hypoxanthine and xanthine are associated with xanthine oxidase deficiency
Lecture, Manual 8 mra
Med1C Villiran – Nucleotide Metabolism Biochem 1b

 Genetic defect or to severe liver damage.

Adenosine Deaminase & Purine Nucleoside Phosphorylase Deficiency

 Meaning no degradation of purines to uric acid


 Adenosine deaminase deficiency = T cell and B cell immunodeficiency. Result: SCID.
 Purine nucleoside phosphorylase deficiency = just T cells (thus, milder)
 Immune dysfunctions appear to result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase
and thereby deplete cells of DNA precursors.

Table (from manual) of Purine disorders


Clinical Disorder Defective Enzyme Nature of Defect Characteristics Inheritance Pattern
Superactive (Vmax)
Resistance to X-linked recessive
Purine
PRPP synthetase feedback inhibition
Gout overproduction and
Low Km for ribose-5-
overexcretion Probably X-linked
phosphate
recessive
HGPRT Partial deficiency
Lesch-Nyhan Same as above PLUS
HGPRT Complete deficiency X-linked recessive
Syndrome self-mutilation. Weird.
Combined T cell and B
Immunodeficiency Adenosine deaminase Severe deficiency cell immunodef,
deoxyadenosinuria
T cell deficiency
(deoxy-)inosinuria
Purine nucleoside
Immunodeficiency Severe deficiency (deoxy-
phosphorylase
)guanoinosinuria Autosomal recessive
hypouricemia
Adenine 2,8-dihydroxyadenine
Renal lithiasis Complete deficiency
phosphoribosyltransferase renal lithiasis
Xanthine renal
Xanthinuria Xanthine oxidase Complete deficiency lithiasis,
hypouricemia

Lecture, Manual 9 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

We can talk about PYRIMIDINES too, I guess


De Novo PYRIMIDINE Nucleotide Biosyn
Step 1: Catalyzed by: Carbamoyl phosphate synthetase II (CPS II)
 This is the committed step
 Enzyme CPS II
o Inhibited by UTP
o Activated by ATP and PRPP
o Cytosolic
o Uses γ-amide group of amine as source of nitrogen

Step 2: formation of carbamoyl aspartate catalyzed by aspartate transcarbamoylase (ATCase)


Step 3: Pyrimidine ring closed by dihydroorotase resulting to dihydroorotate…
Step 4: …oxidized to orotic acid
Step 5: Complete pyrimidine ring converted to orotidine 5’-monophosphate
 PRPP – ribose 5-phosphate donor
 Orotate phosphoribosyl transferase produces OMP with release of pyrophosphate
o Biologically irreversible
Step 6: OMP converted to UMPBy Orotidylate decarboxylase (unsure why this was in ppt, since img in ppt says OMP decarboxylase)
 Deficiency: Orotic aciduria

Lecture, Manual 10 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

Syn of Uridine Triphosphate and Cytidine Triphosphate

Amination of UTP
1. By CTP synthase (synthetase, saw both used)
2. Nitrogen provided by glutamine

Catabolism (Degradation) of Cytosine, Uracil and Thymine


Degradation
Pyrimidine rings can be degraded to highly
soluble structures
 Such as β- alanine & β-aminoisobutyrate

Different Diseases of PYRIMIDINE Metabolism


 Few clinically significant disorders of overproduction of pyrimidine catabolites
o End products are highly water soluble
 Hyperuricemia associated with severe overproduction of PRPP
o Overproduction of pyrimidine nucleotides and increased excretion of ß-alanine
o Since N5, N10 –methylene- tetrahydrofolate is required for thymidylate synthesis, disorders of folate and
vitamin B12 metabolism results in deficiencies of TMP.

Orotic acidurias
 Charac’d by high levels of urinary orotic acid, retarded growth and megaloblastic anemia
 Treat w/uridine and high pyrimidine diet
 One type that accompanies Reye’s syndrome
o Probably consequence of the inability of severely damaged mitochondria to utilize carbamoyl phosphate
o w/c then becomes available for cytosolic overproduction of orotic acid

Orotic Aciduria Type I Type II (Rarer)


Deficient Enzyme Both Only
Orotate phosphoribosyltransferase
Orotidylate decarboxylase Orotidylate decarboxylase

Prevents formation OMG  UMP  UTP w/c  CTP UMP  UTP w/c  CTP
of
Inhibiting Step 2 enzyme of Purine de novo – ATCase, per CTP syn
manual tho I don’t understand why

Deficiency of a Urea Cycle Enzyme results in Excretion of Pyrimidine Precursors (OTCD)


 Excretion of orotic acid, uracil, and uridine accompanies a deficiency in liver mitochondrial ornithine
transcarbamoylase.
Lecture, Manual 11 mra
Med1C Villiran – Nucleotide Metabolism Biochem 1b

 Excess carbamoyl phosphate exits to the cytosol, where it stimulates pyrimidine nucleotide biosyn
 The resulting mild orotic aciduria is increased by high nitrogen containing food.
 Manifest: Protein intolerance and hyperammonemia w/hepatic encephalopathy
Table (from Manual) of Pyrimidine Disorders
Clinical Disorder Defective Enzyme Characteristics Inheritance Pattern
Beta-Aminoisobutyric Transaminase No symptoms; frequent in Autosomal recessive
aciduria Asians
Orotic aciduria, Type I Orotate Orotic acid crystalluria
phosphoribosyltransferase Failure to thrive
and orotidylate Megaloblastic anemia
decarboxylase Immunodef
Tx:Remission w/oral uridine
Orotic aciduria, Type II Orotidylate decarboxylase Orotidunria
Orotic aciduria
Megaloblastic anemia
Tx:Remission w/oral uridine
Ornithine transcarbamoylase Ornithine transcarbamoylase Protein intolerance X-linked recessive
deficiency (OTCD) Hepatic encephalopathy
Mild orotic aciduria

Drugs May Precipitate Orotic Aciduria

 Allopurinol
o alternative substrate for orotate phosphoribosyltransferase
o competes with orotic acid
 Resulting nucleotide product also inhibits orotidylate decarboxylase  orotic aciduria and ortidinuria
 6-Azauridine
o following conversion to 6-azauridylate, also competitively inhibits orotidylate decarboxylase
o enhancing exretion of orotic acid and orotidine

Different Drugs involved in PYRIMIDINE Metabolism – Cancer & Viral Chemotherapy


1. Aminopterine/Amithopterine/Methotrexate
 Inhibits dihydrofolate reductase.(mxn discussed below w/thymidine monophosphate syn)

2. 5 Flurouracil (5 FU)
 An analogue of thymine
 Treatment of solid tumors
o It is converted to the monophosphate nucleotide form via the salvage pathway.
o Eventually, converted to the deoxynucleotide form and binds to thymidylate synthetase  inhibiting the
formation of TMP.
o As a deoxytriphosphate form, it can be incorporated into RNA and inhibits the formation of mature RNA (a
step important in translation)
3. 5-Iodouracil
 Analogue of thymidine
 When incorporated into DNA bonds with C rather than A, thus causing misreading of the strand.

4. Cytosine arabinoside (sugar is replaced to an arabinose)


 Treatment of leukemias.
 Inhibits DNA polymerase in its triphosphate form
 It has a short half life.

Lecture, Manual 12 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

Nucleotides again – Add’l Important rxns


Conversion of Ribonucleotides  DEOXYRibonucleotides
Cells produce dNTPs for DNA syn – enhanced during S(ynthetic) aka “pre-mitotic” phase of cell cycle

Ribonucleotides
 Use as building blocks in RNA synthesis & nucleotide carriers of other compound
 Nucleotides required for DNA syn are 2’ – deoxyribonucleotides,
o w/c are produced from ribonucleoside diphosphate

A. Ribonucleotide reductase (ribonucleoside diphosphate reductase )


 A multi subunit enzyme
o 2 identical B1 subunits and 2 identical B2 subunits
o specific for reduction of nucleoside diphosphates (dADP,dGDP,dCDP, and dUDP )
 2 sulfhydryl groups on the enzyme - immediate donors of H atom needed for the reduction
of the 2’-hydroxyl group — during rxn form a disulfide bond

1.Regeneration of reduced enzyme


 For ribonucleotide reductase to continue to produce deoxyribonucleotides, the disulfide
bond created during production of the 2’-deoxy carbon must be reduced.
 Thioredoxin
o source of reducing equivalents
o peptide coenzyme of ribonucleotide reductase
o contains 2 Cys residues separated by 2 aa’s in peptide chain
o The 2 sulfhydryl groups of thioredoxin donate their hydrogen atoms to ribonucleotide reductase in the
process of forming a disulfide bond.

2. Regeneration of reduced Thioredoxin


 Thioredoxin then must be converted back
to its reduced form in order to continue
its function.
 Necessary equivalents are provided by
NADPH + H
 Catalyzed by Thioredoxin reductase,
protein cofactor

B. Regulation of Deoxyribonucleotide Syn

Ribonucleotide reductase
 Responsible for maintaining a balance supply of the deoxyribonuclotides required for DNA synthesis.
 Note: in add’n to the single active site, there are two allosteric sites on for regulating activity

1. Activity Site: allosteric site for binding of dATP


 inhibits the overall catalytic activity of the enzyme
2. Substrate specificity site: add’l allosteric site for binding of NTP,
 regulates substrate specificity causes
conversion of ribonucleotides 
deoxyribonucleotides as they are
req’d for DNA syn

Lecture, Manual 13 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

Syn of Thymidine Monophosphate from dUMP


dUMP is converted to dTMP by thymidylate synthetase
 w/c utilizes N5, N10 – methylene tetrahydrofolate (FH4 or THF) as methyl donor
o simultaneously oxidized to FH2
 An unusual rxn - in that tetrahydrofolate (THF) contributes
o ONE carbon unit but also TWO hydrogen atoms
o for the pteridine ring
o resulting in the oxidation of THF to dihydrofolate (DHF)

DHF can be reduced to THF by dihydrofolate reductase


 a reductive syn
 inhibited in the presence of drugs such as methotrexate.
o Competitively inhibits DHF reductase
o by decreasing the supply of THF, these folate analogs not only inhibit purine synthesis, but by preventing
methylation of dUMP to dTMP, they also lower the cellular concentration of this essential component of DNA
o Ex: Aminopterine/Amithopterine
 Inhibitors of Thymidylate synthetase include thymine analogs
o such as 5- fluoracil
o DNA synthesis is therefore inhibited and cell growth slowed
 Trimethoprim (in Bactrim, Cotrimoxazole)
o Similar mxn
o Acts more on bacterial cells a good antibiotic
o Generally used in combo with sulfamethoxazole (sulfonamide)
 A paraaminobezoic acid analogue (PABA)
 Constituent of folic aicd

Lecture, Manual 14 mra


Med1C Villiran – Nucleotide Metabolism Biochem 1b

 prevent folic acid syn in bacterial cell (but not man)  inhibiting TMP syn and DNA rep in bacterial
cells

Lecture, Manual 15 mra