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Background

• Nucleotides (and other compounds derived

from nucleotides) have diverse but critical
METABOLISM OF PURINE AND functions in cellular metabolism (all cells)
PYRIMIDINE NUCLEOTIDES
1. Parent compounds of nucleic acids
– Deoxyribonucleotides in DNA

NIGANI WILLIE
– Ribonucleotides in RNA
DISCIPLINE OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
DIVISION OF BASIC MEDICAL SCIENCES
SCHOOL OF MEDICINE AND HEALTH SCIENCES
UNIVERSITY OF PAPUA NEW GUINEA
TAURAMA CAMPUS

2. Energy Metabolism 3. Structural components of various enzyme

– ATP (generated via oxidative or substrate level cofactors
phosphorylation)
– NAD+ / NADH
– Energy currency for metabolic transactions
– NADP+ / NADPH
(stored chemical energy for cells)
– phosphorylation agent involved in muscle – FAD+ / FADH
contraction, active transport and maintenance – coenzyme A
of ion gradients
– phosphate donor for generating other
nucleotides

Nitrogenous Bases of Nucleic Acids

4. Metabolic intermediates of numerous
metabolic pathways in cells • Nitrogenous bases in nucleic acids are
– Control of coronary blood flow – Adenosine derivatives of two parent compounds:
– Platelet aggregation (blood coagulation) – ADP
– Capping of mRNA, signal transduction,
1. Purine
microtubule formation - GTP
– UDP-glucose – glycogen and glycoprotein
synthesis 2. Pyrimidine
– GDP-mannose, GDP-fucose, UDP-galactose, CMP-
sialic acid – glycoprotein synthesis
– CTP – phospholipids metabolism

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What are Purines and Pyrimidines? • Are hydrophobic (relatively insoluble in water
at near-neutral pH)
• Purines and pyrimidines are nitrogen- • However at acidic or alkaline pH, the bases
containing compounds become charged and their solubility in water
increases
• have a heterocyclic structure
• Hydrophobic stacking interactions between
• they are major components (bases) of bases are important in nucleic acid structure
nucleotides (and nucleosides)
• have a variety of chemical properties that
• Weakly basic (alkaline) compounds and are affect the structure and ultimately the
thus called bases function of nucleic acids

PURINES Other purine compounds also encountered are:

• Major purine bases: • Hypoxanthine
adenine guanine • Xanthine

• Caffeine (in coffee)

• Theophylline (in tea)

• Theobromine (in chocolate)

PURINE METABOLISM
• Metabolism involving purine nucleotides can
occur in three ways:
1. By De novo synthesis pathways

2. By Breakdown of dietary nucleic acids and

Degradation of purine nucleotides

3. By Salvage (Recycling) pathways

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De novo synthesis of Purine Nucleotides De novo synthesis of Purine Nucleotides

• De novo synthesis begins with the formation • To form Inosine 5’-monophospate (IMP),
of 5-phosphoribosyl-1-pyrophosphate (PRPP) nucleotide with closed purine ring, PRPP
from Ribose 5-phosphate undergoes 10 enzyme-catalysed reactions

Ribose 5-phosphate PRPP

5,10-
MethylH4folate,
(10 steps)
PRPP synthetase Aspartate, Glycine,
Glutamine, CO2,
10-FormylH4folate

PRPP IMP

CO2
• Compounds from other pathways (5,10- Glycine
Aspartate
MethylH4folate, Aspartate, Glycine,
Glutamine, CO2, 10-FormylH4folate) are used
in the formation of the purine ring during the
biosynthesis of IMP
N5,N10-methenyl
H4folate

* (Ribose 5-phosphate is an end-product of the

Pentose Phosphate Pathway, also called the
Hexose Monophosphate (HMP) Shunt)
N10-formyl
H4folate amide nitrogen
of Glutamine

IMP D-ribose5-phosphate
PRPP-synthetase

Adenylosuccinate synthetase IMP dehydrogenase PRPP

PRPP-amidotransferase

IMP
Adenylosuccinate XMP
GMP synthetase AMP GMP
Adenylosuccinase

ADP GDP
AMP GMP
ATP GTP

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• Free nitrogenous bases are NOT used as Examples of Purine Nucleotides

intermediates in de novo synthesis
• Adenosine 5’-monophosphate (AMP)
• The purine ring is built ONE-ATOM-AT-A-TIME • Adenosine 5’-diphosphate (ADP)

• De novo synthesis is expensive in terms of ATP • Adenosine 5’-triphosphate (ATP)

usage
• Deoxyadenosine 5’-monophosphate (dAMP)
• Regulation of purine nucleotide is regulated
by feedback inhibition i.e. inhibition of certain • Deoxyadenosine 5’-diphosphate (dADP)
enzymes that catalyse the reactions in the • Deoxyadenosine 5’-triphosphate (dATP)
pathway by end products

Degradation of Nucleic Acids And Nucleotides

• Guanosine 5’-monophosphate (GMP) 1. Nucleic acids, RNA and DNA, can be

hydrolysed to free nucleotides during
• Guanosine 5’-diphosphate (GDP) digestion or cell turnover by nucleases
• Guanosine 5’-triphosphate (GTP)

• Deoxyguanosine 5’-monophosphate (dGMP) hydrolysis

nuclease
• Deoxyguanosine 5’-diphosphate (dGDP)
• Deoxyguanosine 5’-triphosphate (dGTP)

2. Nucleotides can be degraded to nucleosides 3. Purine nucleosides can be degraded to purine

bases by purine nucleoside phosphorylases
by nucleotidases
nucleoside purine base
purine nucleoside phosphorylase
G G
nucleotidase
S

P P
Pi
S

Example: GMP Guanosine Example: Guanosine Guanine

Pi Ribose
Pi phosphate

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4. Purine bases can be metabolised to Uric Acid 4. Purine bases can be metabolised to Uric Acid
* Degradation of adenine (or its corresponding
nucleoside adenosine) to uric acid requires
* Guanine degradation to Uric Acid involves more reactions than guanine (and guanosine)
2 steps adenosine
Adenosine deaminase
Inosine
Pi Pi
xanthine purine purine
guanase oxidase nucleoside nucleoside
Guanine Xanthine Uric Acid phosphorylase
Ribose
phosphorylase
Ribose
phosphate phosphate
xanthine
Adenine Hypoxanthine oxidase
Xanthine
xanthine
xanthine oxidase
oxidase

Dihydroxyadenine Uric Acid

Summary of Uric Acid Formation

Salvage Pathways of Purine Nucleotides
Nucleic Acids
• The products of nucleic acid and nucleotide
nuclease
degradation (nitrogenous bases and nucleosides)
Nucleotides
can be used to reform nucleotides
nucleotidase

Nucleosides
• Two important enzymes involved in recycling are:
purine nucleoside phosphorylase
1) Hypoxanthine-guanine phosphorybosyl
Purine Bases
transferase (HGPRTase)
xanthine oxidase
2) Adenine phosphorybosyl transferase
Uric Acid
(APRTase)

D-ribose5-phosphate
HGPRTase PRPP-synthetase

PRPP
PRPP-amidotransferase
hypoxanthine + PRPP IMP + PPi
AMP AMP
IMP GMP
APRTase HGPRTase HGPRTas
deaminase
e
guanine + PRPP GMP + PPi 5’-nucleotidase 5’-nucleotidase 5’-nucleotidase

Adenosine
deaminase
Adenosine Inosine Guanosine
Purine Purine Purine
nucleoside nucleoside nucleoside
phosphorylase phosphorylase phosphorylase
APRTase Adenine Hypoxanthine Guanine

xanthine oxidase xanthine oxidase guanase

adenine + PRPP AMP + PPi

Dihydroxyadenine Xanthine
xanthine oxidase

Uric Acid
*Salvage Pathways in Green

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What is URATE?

• Urate is the ionized form of uric acid

• At physiological pH, uric acid is almost

completely ionised to urate

• Urate ions form sodium urate (or

monosodium urate), the principal form of
urate in plasma or serum

Plasma Urate
• Plasma concentration of urate varies depending
on different physiological factors:

1. Sex – plasma [urate] reference range is higher

in males (0.12-0.42 mmol/L) than in females
(0.12-0.36 mmol/L)

2. Obesity – plasma [urate] tends to be higher in

the obese

3. Social class – the more affluent social classes tend Clinical problems
to have higher plasma [urate]
Gout
4. Diet – plasma [urate] rises in individuals taking a
high protein diet, that is, a diet that is also rich in • Gout is characterised by:
nucleic acids, and those with a high alcohol – Hyperuricaemia
consumption – Recurrent acute arthritis
5. Genetic factors; defects in: – Acute gout is triggered by tissue deposition of
– enzymes in purine metabolism sodium urate crystals causing an inflammatory
response
– proteins involved in urate excretion affect plasma [urate]
– other pathways may result in the increase of precursors – In chronic situation, tophaceous deposits of
or intermediates for de novo purine synthesis sodium urate may form in the tissues

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Gout may occur due to: • Gout is exacerbated by alcohol

– Ethanol increases turnover of ATP and urate
• Increase in PRPP synthetase activity production
• Partial deficiency of HGPRTase activity – Excess ethanol may cause the accumulation of
organic acids which compete with the tubular
• defects in other pathways secretion of uric acid
– urate excretion pathway – Ethanol also causes dehydration which causes
super-saturation that could lead to sodium urate
– pathways that provide precursors or regulate the crystal formation
level of intermediates for de novo synthesis
• Disorders such as ethanol intoxication, diabetic
 for example: Glucose-6-phosphatase deficiency leads to
ketoacidosis and starvation lead to elevations of
excessive production of ribose-5-phosphate via glucose
6-phosphate dehydrogenase (G6PD). Ribose-5-
-hydroxybutyric acid and acetoacetic acid and
phosphate is a precursor for the de novo synthesis of cause hyperuricaemia
purine nucleotides, nucleosides and bases

Lesch-Nyhan syndrome Acute Uric Acid Nephropathy

• Severe or complete deficiency of HGPRTase • High levels of uric acid in the urine
resulting in: (hyperuricosuria) can cause impairment of
renal function (renal insufficiency)
– excessive uric acid production; hyperuricaemia
– neurological problems • Acute Uric Acid Nephropathy is caused by
• Although research suggest defects in purine deposition of uric acid crystals within the
metabolism cause neurological problems because kidney intersitium and tubules
brain cells rely mostly on salvage pathways for
their nucleotide generation, the exact mechanisms
remain unclear
• This may lead to complete or partial
obstruction of collecting ducts, pelvis or ureter

PYRIMIDINES PYRIMIDINE METABOLISM

• Metabolism of pyrimidine nucleotides also

involves:

1. De novo synthesis pathways

2. Breakdown of dietary nucleic acids and
Degradation of purine nucleotides
3. Salvage (Recycling) pathways

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De novo synthesis of Pyrimidine Nucleotides

• Multifunctional proteins CAD and UMP
• In the de novo synthesis of pyrimidine synthetase are involved in pyrimidine de novo
synthesis
nucleotides, the six-membered ring called
orotate is formed separately from the other 1. CAD – trifunctional
nucleotide components
– 3 enzymatic functions:
• PRPP is then attached to orotate 1. carbamoyl phosphate synthetase II
• Precursors: Glutamine, Aspartate, ATP, CO2 2. aspartate carbamoyl transferase
3. dihydroorotase

De novo synthesis of Pyrimidine Nucleotides

Glutamine CO2 2ATP

• Dihydroorotate is converted to OROTATE by a
Carbamoyl phosphate synthetase II
dihydroorotase dehydrogenase, a
Carbamoyl phosphate
mitochondrial enzyme
Aspartate
CAD Aspartate carbamoyl synthetase
• Orotate is the first completed pyrimidine ring
N-carbamoyl aspartate in the de novo synthesis
Dihydroorotase

Dihydroorotate

2. UMP synthetase – bifunctional

Dihydroorotate
– 2 enzymatic functions:
NAD+

Dihydroorotase dehydrogenase
1. orotate phosphoribosyl transferase
NADH + H+

2. OMP decarboxylase
OROTATE

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OMP
OROTATE
OMP decarboxylase
PRPP
Orotate phosphoribosyl transferase UMP CMP

UMP synthetase Orotidine 5’-monophosphate (OMP)

UDP CDP
OMP decarboxylase Glutamate ADP + Pi
Glutamine ATP

UTP CTP
CTP Synthase
Uridine 5’-monophosphate (UMP)

Glutamine CO2 2ATP

• PRPP is also required in the de no synthesis of
pyrimidine nucleotides
Carbamoyl phosphate UTP CTP
• PRPP + orotate  OMP

• OMP is then converted to UMP N-carbamoyl aspartate UDP CDP

• UMP is converted to UDP and UTP Dihydroorotate UMP CMP

• UTP is converted to CTP

OROTATE OMP

• De novo synthesis of pyrimine nucleotides is Ribonucleotide = nitrogenous base +

energy expensive in terms of ATP usage ribose sugar + phosphate

• Regulation of pyrimidine nucleotide is Ribonucleotide = nucleoside + phosphate

achieved by feedback inhibition i.e. inhibition
of enzymes that catalyse the reactions in the
pathway by end products Ribonucleotide = nucleoside 5’-monophosphate
= nucleoside 5’-diphosphate
• Thymidine nucleotide (dTMP) is derived from = nucleoside 5’-triphosphate
dCDP and dUMP from a separate pathway

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Ribonucleotide = nitrogenous base + sugar + Ribonucleotide = nitrogenous base + sugar +

phosphate phosphate

Ribonucleotide = nucleoside + phosphate Ribonucleotide = nucleoside + phosphate

Ribonucleotide Ribonucleotide
e.g. Adenosine 5’-monophosphate e.g. Guanosine 5’-monophosphate
Adenosine 5’-diphosphate Guanosine 5’-diphosphate
Adenosine 5’-triphosphate Guanosine 5’-triphosphate

Ribonucleotide = nitrogenous base + sugar + Ribonucleotide = nitrogenous base + sugar +

phosphate phosphate

Ribonucleotide = nucleoside + phosphate Ribonucleotide = nucleoside + phosphate

Ribonucleotide Ribonucleotide
e.g. Cytidine 5’-monophosphate e.g. Uridine 5’-monophosphate
Cytidine 5’-diphosphate Uridine 5’-diphosphate
Cytidine 5’-triphosphate Uridine 5’-triphosphate

FORMATION OF DEOXYRIBONUCLEOTIDES Formation of Deoxyribonucleotides

Ribonucleotide • Reduction of the 2′-hydroxyl of purine and

pyrimidine ribonucleotides, catalyzed by the
ribonucleotide reductase complex, forms
Ribonucleotide reductase deoxyribonucleoside diphosphates (dNDPs)
complex

• The enzyme complex is active only when cells

are actively synthesizing DNA.
Deoxyribonucleotide

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Formation of Deoxyribonucleotides Ribonucleotide Reductase Reaction

Requires:
Ribonucleoside 5’-diphosphates
1) Nucleoside 5’-triphosphates
– ATP, GTP, CTP, TTP
Ribonucleotide reductase
complex
2) Mg2+

3) Glutathione (or Thioredoxin)

2’-deoxyribonucleoside 5’-diphosphate

Ribonucleotide Reductase Reaction Ribonucleotide Reductase Reaction

Requires:
P P B P P B

4) Enzyme for coupling reaction S S

OH OH OH H
– Glutathione reductase
– Thioredoxin reductase glutathione glutathione

5) Reducing equivalent
NADP+ NADPH + H+
– NADPH Glutathione Reductase

Ribonucleotide reductase reaction TMP = dTMP

ADP dADP TMP = thymidine 5’monophosphate

GDP dGDP = deoxythymidine 5’- monophosphate

CDP dCDP
= 2’-deoxythymidine 5’- monophosphate
UDP dUDP
= deoxythymidylate

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Thymidine 5’monophosphate (TMP) Formation TMP Synthase Reaction

dUMP dTMP
CDP dCDP dCMP

TMP synthase
UDP dUDP dUMP
P S P S
OH H OH H
5,10-MethylH4folate dihydrofolate
TMP synthase

TMP
*TMP synthase is also called Thymidylate synthase

TMP Synthase reaction Degradation of Pyrimidine Nucleotides

RNA
• 5, 10-Methylene-tetrahydrofolate is used as a nucleases nucleases
methyl donor in the TMP synthase reaction
CMP UMP
• 5, 10-Methylene-tetrahydrofolate is derived
phosphatases phosphatases
from folate (a B vitamin) pyrimidine
nucleoside
deaminase
• Deficiency of folate (and B12) result in TMP Cytidine Uridine
deficiency and affects nucleic acid nucleoside
phosphorylase
biosynthesis and cell division
Uracil

Degradation of Pyrimidine Nucleotides Degradation of Pyrimidine Nucleotides

DNA Uracil
nucleases nucleases dihydropyrimdine
dehydrogenase

dUMP dCMP dTMP Dihydrouracil

phosphatases phosphatases phosphatases dihydropyrimidinase

dUridine dCytidine dThymidine -Ureidopropionase

nucleoside nucleoside ureidopropionase
phosphorylase phosphorylase

Uracil Thymine -Alanine

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Degradation of Pyrimidine Nucleotides Degradation of Pyrimidine Nucleotides

Thymine • Since the end products of pyrimidine
dihydropyrimdine catabolism (degradation) are highly water-
dehydrogenase
soluble, pyrimidine overproduction results in
Dihydrothymine few clinical problems
dihydropyrimidinase

• In hyperuricaemia that results from severe

-Ureidoisobutyrate overproduction of PRPP there is also an
ureidopropionase overproduction of pyrimidine nucleotides and
therefore increased excretion of β-alanine
-Aminoisobutyrate

Salvaging of Pyrimidine Bases Study Questions

1. What are some functions of nucleotides?
2. What are purines? Name some common purines
Pyrimidine Pyrimidine nucleoside 3. What are pyrimidines? Name pyrimidines
+ PRPP + PPi
Base 5’-monophosphate 4. List some common purine nucleotides and their corresponding
nucleosides
5. List some common pyrimidine nucleotides and their
corresponding nucleosides
Orotate OMP
6. What is de novo synthesis of purines?
7. How does the salvaging of purines occur? Name the two
Uracil UDP enzymes required for salvaging purines?
8. Describe the degradation of nucleic acids and purine
nucleotides? List the types of enzymes involved in the
Thymine dTMP degradation pathway

8. What is uric acid? REFERENCES

9. What is hyperuricaemia? How is hyperuricaemia caused?
Outline the factors that affect blood uric acid levels • Murray RK, Granner DK, Mayes, Rodwell VW. Harper’s Biochemistry 24th
Edition 1996
10. What is gout? Causes of gout?
11. What is the deficiency leads to Lesch-Nyhan syndrome? • Koolman J and Roehm KH. Colour Atlas of Biochemistry 2nd Edition 2005
12. What is uricosuria? What causes Acute Uric Acid Nephropathy?
13. How is the de novo synthesis of Purine nucleotides different • Devlin, TM (editor). Textbook of Biochemistry with Clinical Correlations
from that of Pyrimidine nucleotides 6th edition, 2006, John Wiley & Sons Inc.
14. What is the significance of the ribonucleotide reductase
• Nelson DL and Cox MM. Lehninger Principles of Biochemistry 3rd Edition,
reaction? Name the cofactors required in this reaction 2000, Worth Publishers, New York.
15. What is the significance of the thymidylate synthase (TMP
synthase) reaction? What is the cofactor required in this • Gaw A, Cowan RA, O’Reily DSt.J, Stewart MJ and Shepherd. Clinical
reaction? Biochemistry (An Illustrated Text), 1995, Churchill Livingstone
16. What are the end products of pyrimidine degradation?
17. What is orotic aciduria? • Beckett, G., Walker, S., Rae, P and Ashley, P. Clinical Biochemistry, 7th
Edition. 2005. Blackwell Publishing, Oxford, UK

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