1. Purine Metabolism/Diseases
Lecture 29
Purine Metabolism/Diseases
Raymond B. Birge, PhD

2. Nucleotides - key roles in cellular processes:
1. Activated precursors of RNA and DNA
2. Adenine nucleotides are components of the major
co-enzymes, NAD, NADP, FMN, FAD, and CoA
3. Nucleotide derivatives are activated intermediates in
biosynthetic processes (UDP-glucose, SAM)
4. Serve as metabolic regulators (e.g cAMP and
the activation of cell signaling).
5. Serve as major currency of energy in all cells
(ATP and GTP).
6. Several metabolic diseases have their etiology in nucleotide
metabolism.

3. Purine metabolism (Overview)
1. Nomenclature/nucleotide structure
2. De novo synthesis pathways
3. Re-utilization (salvage) pathways
4. Degradation pathways
5. Metabolic diseases of purine
metabolism (Gout, Lesch-Nyhan,
SCID)
Suggested reading: Lippencott’s Chapter 22

4. Numbering of sugar carbons with ‘prime’

5. Adenosine monophosphate
Nomenclature
Adenine
BASE
Adenosine
NUCLEOSIDE
Adenosine monophosphate
(AMP)
NUCLEOTIDE
Another name for nucleotide is Adenylic Acid/Adenylate 5

6. Active forms of nucleotides: di-and tri-phosphates
GMP + ATP
GDP + ADP
Nucleoside Monophosphate Kinase
(i)
To get active form of nucleotindes
Specific reaction for cytidine MP
General for generating the triphosphate
XDP + YTP
XTP + YDP
Nucleoside Diphosphate Kinase
(ii)

7. Why are nucleosides and nucleotides important For biochemists?
Purine binding proteins (“the purine proteome”) comprise a family of 3-4,000
Proteins and as much as 50% of all druggable targets in biology.
Kinases
Helicases
Reductases
Transferases
Synthetases
Dehydrogenases
Chaperones
Metabolic Enzymes
DNA and RNA processing
Etc

8. Common Purine Bases H= 6 oxy purine A= 6 amino purine
Hypoxanthine O
Xanthine
Guanine
NH2
Adenine O
H= 6 oxy purine
X= 2,6 dioxy purine
A= 6 amino purine
G= 2 amino, 6-oxy purine

9. Nucleoside Function in extracellular signal transduction
Adenosine nucleoside-increased
during ATP degradation.
Released in cells when there
is low O2 concentration
Binds to purinogenic receptors
A1, A2A, A2B, A3
Slows the heart down, at the same
time increases capillary dilation
Caffeine is a adenine derivative, and
antagonizes the effects of adenine.

12. Cyclic nucleotides are important mediators for
Intracellular signal transduction
ATP
cAMP
PKA
phosphorylase
kinase
glycogen
synthase P
10-10 M

13. Two pathways to generate purines and pyrimidines
1. DE NOVO BIOSYNTHETIC PATHWAYS
(building the bases from non-purine molecules)
2. SALVAGE PATHWAYS
(the reutilization of bases from dietary
or catabolic sources)

14. De novo biosynthesis of purines (A and G):
activation of ribose-phosphate
Ribose phosphate pyrophospho-
Kinase (PRPP synthase)
Pentose phosphate
pathway
IMP, AMP, GMP
In unit 4, energy metabolism, Dr. Michael Lea discussed the Pentose Pathway

15. Major regulatory step in purine biosynthesis:
PRPP to 5-Phosphoribosyl-1-amine *
Glutamate
Glutamine
PPi
PRPP
Amidophosphoribosyl
transferase
Inhibited by products IMP, AMP, and GMP.
FEEDBACK INHIBITION

16. Purine biosynthesis intermediates
Carboxylation unusual in not using biotin.

17. Purines: where do the atoms come from?
Key: Glycine
1 C unit of N10 f-TetraHydroFolic acid (sulfonamides; methotrexate)
Glutamine
Asparate 17

18. Amino acids utilized in purine biosynthesis
Carboxylation unusual in not using biotin.
Inosine monophosphate is the precursor for both AMP and GMP

19. AMP & GMP synthesis
IMP 19

20. Hypoxanthine to Adenine/Guanine.
Xanthine-XMP
Hypoxanthine-IMP
(N source)
NH2
Aspartate
Adenine O
(N source)
Glutamine
NH2
Guanine
The common mechanistic theme for the conversion to A and G is the
conversion of a carbonyl oxygen to an amino group

21. Regulation of purine de novo biosynthesis: classic negative feedback
Ribose
5-phosphate
PRPP
Phosphoribosyl
amine
IMP
AMP
GMP
Inhibited by AMP
Inhibited by GMP
Inhibited by IMP,
AMP, and GMP

22. Salvage pathways: re-utilizate purinesO P OH
CH2
PPi +
Base
(Adenine
or Guanine) A
PRPP + Adenine
Adenylate/AMP
PRPP + Guanine
Guanylate/GMP
A-PRT
HG-PRT
There are 2 salvage enzymes with different specificities:
1. Adenine phosphoribosyl transferase (APRT)
2. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

23. Nucleoside monophosphates
Stages of nucleotide metabolism
Endonuclease
Phosphodiesterase
Nucleoside monophosphates
(Mononucleosides)
Nucleoside
triphosphate
Nucleic Acid
Synthesis

24. Nucleoside monophosphates
Endonuclease
Nucleic Acid
Synthesis
Nucleoside
triphosphate
Phosphodiesterase
Nucleoside monophosphates
(Mononucleosides)
H20
Nucleotidases
PPi Pi
ADP
Phosphoribosyl
transferases
Nucleoside kinase
Nucleosides
ATP Pi
Phosphorylases
PRPP
Ribose-1-P
Nucleobases (A, G)
Uric Acid (purines)

25. Adenosine monophosphate
Getting Back to ‘Basics’
Nucleotidase
Phosphorylase
Adenine
Adenosine monophosphate
(AMP)
Adenosine
NUCLEOTIDE
NUCLEOSIDE
BASE

26. Purines in humans are degraded to urate
(ADA)

27. Gouty Arthritis The Gout By Royal Authority James Gilray 1799
Classification of Hyperuricemia
Overproduction of urate • Primary idiopathic hyperuricemia • Hypoxanthine-guanine phosphoribosyl-transferase deficiency • Phosphoribosylpyrophosphate synthetase overactivity • Hemolytic processes • Lymphoproliferative disease • Myeloproliferative disease • Polycythemia vera • Psoriasis (severe) • Paget's disease • Rhabdomyolysis • Exercise • Alcohol • Obesity • Purine-rich diet Decreased excretion of uric acid • Primary idiopathic hyperuricemia • Renal insufficiency • Polycystic kidney disease • Diabetes insipidus • Hypertension • Acidosis --Lactic acidosis --Diabetic ketoacidosis • Down syndrome • Starvation ketosis • Berylliosis • Sarcoidosis • Lead intoxication • Hyperparathyroidism • Hypothyroidism • Toxemia of pregnancy • Bartter's syndrome
Decreased excretion of uric acid (continued) • Drug ingestion --Salicylates (less than 2 g per day) --Diuretics --Alcohol --Levodopa-carbidopa (Sinemet) --Ethambutol (Myambutol) --Pyrazinamide --Nicotinic acid (niacin; Nicolar) --Cyclosporine (Sandimmune)  
Combined mechanism • Glucose-6-phosphate dehydrogenase deficiency • Fructose-1-phosphate aldolase deficiency • Alcohol • Shock
By Royal Authority
‘King George IV’
George Cruickshank
19th C.

28. Some famous people who had gout
Henry VIII
Kublai Khan
Nostradamus
John Milton
Isaac Newton
Frederick the Great
John Hancock
Thomas Jefferson
Benjamin Franklin
David Wells
Colchisine exerts its effect by inhibiting the phagocytosis of uric acid and blocking the release of chemotactic factor.

29. Gout results from HYPERURICEMIA
Decreased URIC ACID excretion: 80% of gout -
ideopathic, renal disease, diabetes insipidus,
hypertension, Downs syndrome, many others
Increased URIC ACID production: 20% of gout –
PRPP synthase overactivity, hemolytic diseases,
lymphoproliferative diseases, may others
HGPRT deficiency (Lesch Nyhan Syndrome),
exacerbated by alcohol, purine rich diet, obesity

30. PRPP Gout from increased uric acid production
1. Lost regulation of PRPP Synthase & PRPP Amidotransferase
Ribose
5-phosphate
Phosphoribosyl
amine
PRPP
purines
Inhibited by IMP,
AMP, and GMP
Leads to net increase in biosynthetic/degradation pathways!!
(From slide #18)

31. Gout from increased uric acid production
2. Defects in salvage pathway lead to increased PRPP & Guanine
PRPP + Guanine
GMP
[HG-PRT]
Leads to net increase in biosynthetic/degradation pathways!!

32. GOUT Treatment Guanine Xanthine Hypoxanthine Urate Allopurinol:
xanthine oxidase
People with high Uric acid levels may or may not have gout. But all people with Gout have
NaUrate crystals in PMLs
Allopurinol:
a. decrease urate
b. increase xanthine &
hypoxanthine
c. decrease PRPP

33. Initiate urate lowering therapy !!
Tophi at
helix of ear
1st MTP joint
(75%) in first metatarsophalangeal joint (big toe: podagra),
-tophi occur approximately 10 years after first attack -tophi seen in synovium, tendons, bursae and subcutaneous tissue -aspiration demonstrates uric acid crystals and inflammatory cells -incidence of tophi has been greatly decreased by the advent of medications to lower uric acid
Synovial fluid: -monosodium urate crystals (intracellular)-needle-shaped crystals that are negatively birefringent under polarized light-diagnostic for gout -increased leukocytes: >15,000/cc WBCs, primarily PMNs C. Tophi aspirate: monosodium urate crystals-diagnostic
Only 5% of persons with hyperuricemia develop gouty arthritis B. 7% of persons will have only one arthritic attack C. 60% will have recurrence within one year D. With prolonged disease, frequency of attacks increases and may even become chronic D. Renal dysfunction seen in 90% of persons with gouty arthritis however, severe renal dysfunction is uncommon. Renal dysfunction may not be due to hyperuricemia, but rather may be due to other concomitant diseases, e.g. HTN, diabetes
normal: males 3-8mg/dl females 1.5-6mg/dl ("normal" values may vary between laboratories)
Large tophaceous
deposits
surrounding joint
Initiate urate lowering
therapy !!

34. Clinical Significance of Purine metabolism
ID: A 56 year old obese white male comes to his family doctor.
Chief Complaint: ‘My big toe hurts like !*?!#*!?!!!’
History Present Illness: Pain began during the night after an episode of binge
eating and drinking.
Past Medical History: Significant for removal of kidney stones last year.
Current Health/Risk Factors: He admits to being an avid meat eater and
drinks beer every night.
Physical Exam: fever, right metatarsophalangial (MTP) joint red, hot and
swollen; painful to motion; subcutaneous deposits in helix of left ear
Pathology: Synovial fluid aspiration shows negatively birefringent,
needle-shaped crystals.
alcohol metabolism produces net adenosine triphosphate degradation to adenosine monophosphate, which is subsequently converted to uric acid. In addition, lactate generated via alcohol consumption increases proximal tubular urate re-absorption while interfering with urinary urate secretion.[36,37] Chronic heavy use of alcohol also has the potential to inhibit conversion of the pro-drug allopurinol to its active metabolite oxypurinol.[38]
Beer contains purines

35. Classification of Hyperuricemia
Overproduction of urate • Primary idiopathic hyperuricemia • Hypoxanthine-guanine phosphoribosyl-transferase deficiency • Phosphoribosylpyrophosphate synthetase overactivity • Hemolytic processes • Lymphoproliferative disease • Myeloproliferative disease • Polycythemia vera • Psoriasis (severe) • Paget's disease • Rhabdomyolysis • Exercise • Alcohol • Obesity • Purine-rich diet Decreased excretion of uric acid • Primary idiopathic hyperuricemia • Renal insufficiency • Polycystic kidney disease • Diabetes insipidus • Hypertension • Acidosis --Lactic acidosis --Diabetic ketoacidosis • Down syndrome • Starvation ketosis • Berylliosis • Sarcoidosis • Lead intoxication • Hyperparathyroidism • Hypothyroidism • Toxemia of pregnancy • Bartter's syndrome
Decreased excretion of uric acid (continued) • Drug ingestion --Salicylates (less than 2 g per day) --Diuretics --Alcohol --Levodopa-carbidopa (Sinemet) --Ethambutol (Myambutol) --Pyrazinamide --Nicotinic acid (niacin; Nicolar) --Cyclosporine (Sandimmune)  
Combined mechanism • Glucose-6-phosphate dehydrogenase deficiency • Fructose-1-phosphate aldolase deficiency • Alcohol • Shock 35

36. Lesch-Nyhan Syndrome Severe HGPRT deficiency: decreased IMP&GMP
X-linked recessive
Severe HGPRT deficiency:
decreased IMP&GMP
increased PRPP & de novo purine p’way
Hyperuremia: gouty arthritis, kidney stones, tophi
Neurologic disability: spasticity, hyperreflexia
Behavioral problems: cognitive dysfunction,
aggression, self-injury 36

37. Severe Combined Immunodeficiency Syndrome
SCID
Severe Combined Immunodeficiency Syndrome
Autosomal recessive disorder
Mutations in ADA*
Infants subject to bacterial,
candidiasis, viral, protazoal
infections
Both T and B cells reduced
(dATP is toxic)
1995-AdV expressing ADA was
successfully employed as gene
therapy strategy
AMP
Adenosine
Inosine
H20 Pi
NH3
Hypoxanthine
Nucleotidase
Adenosine deaminase*
infusion of autologous CD34+ cells from bone marrow that had been transduced with a viral vector carrying the ADA gene.; 10 out of ten children doing well after mean survivial of 4 yrs (1.8-8).

38. Adenosine Deaminase (ADA) deficiency
Urate
(From slide #19)

39. Bottom Line
Recognize names and structures of purines/nomenclature of NMPs:
Adenosine, Guanine, Hypoxanthine, Xanthine
Name the precursors of atoms in the purine ring:
Gln (N); Gly (C, N); N10-fTHF (C ); HCO3- (CO); Asp (N)
Recognize the regulated reactions:
PRPP synthase: IMP, AMP, GMP
Gln:PRPP amidotransferase: IMP, AMP, GMP; PRPP
IMP AMP: AMP
IMP GMP: GMP
Explain the cause of SCIDS
Make differential diagnosis of gouty arthritis and
Lesch-Nyhan Syndrome
Explain mechanisms of the following treatments:
sulfaonamides, methotrexate, allopurinol

40. Summary and Take-Home Points
Identify basic structures of purines, nucleosides, and
nucleotides.
2. Identify key relationships between glucose metabolism
and purine biosynthesis.
3. Knowledge of how amino acids are used in AMP and
GMP biosynthesis.
4. Understand degradation pathways of purines and
their relationship to uric acid metabolism and gout

41. Integrative Thought Question
Ribonucleotides and Deoxyribonucleotides are essential for all cells,
and represent key convergent points in energy metabolism.
Provide specific examples in which purine metabolism can be linked
to metabolism of
1). Glucose
2). Lipids
3). Amino Acids
4). Ammonium