Transcript No Slide Title

Lecture 29
Purine Metabolism/Diseases
Raymond B. Birge, PhD
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.
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
Nomenclature
Adenine
BASE
Adenosine
Adenosine monophosphate
(AMP)
NUCLEOSIDE
NUCLEOTIDE
Active forms of nucleotides:
di-and tri-phosphates
Nucleoside Monophosphate Kinase
(i)
GMP + ATP
(ii)
Nucleoside Diphosphate Kinase
XDP + YTP
GDP + ADP
XTP + YDP
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
Common Purine Bases
O
NH2
Adenine
Hypoxanthine
O
O
O
NH2
Xanthine
H= 6 oxy purine
X= 2,6 dioxy purine
Guanine
Guanine
A= 6 amino purine
G= 2 amino, 6-oxy purine
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.
Cyclic nucleotides are important mediators for
Intracellular signal transduction
10-10 M
cAMP
ATP
PKA
P
phosphorylase
kinase
P
phosphorylase
P
glycogen
synthase
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)
De novo biosynthesis of purines (A and G):
activation of ribose-phosphate
Pentose phosphate
pathway
IMP, AMP, GMP
Ribose phosphate pyrophosphoKinase (PRPP synthase)
Major regulatory step in purine biosynthesis:
PRPP to 5-Phosphoribosyl-1-amine
Glutamine Glutamate
PRPP
*
PPi
Amidophosphoribosyl
transferase
Inhibited by products IMP, AMP, and GMP.
FEEDBACK INHIBITION
Purine biosynthesis intermediates
Purines: where do the atoms come from?
Key: Glycine
1 C unit of N10 f-TetraHydroFolic acid (sulfonamides; methotrexate)
Glutamine
Asparate
Amino acids utilized in purine biosynthesis
Inosine monophosphate is the precursor for both AMP and GMP
AMP & GMP synthesis
IMP
Hypoxanthine to Adenine/Guanine.
O
NH2
(N source)
Aspartate
Hypoxanthine-IMP
Adenine
O
O
(N source)
Glutamine
O
Xanthine-XMP
NH2
Guanine
The common mechanistic theme for the conversion to A and G is the
conversion of a carbonyl oxygen to an amino group
Regulation of purine de novo biosynthesis:
classic negative feedback
Ribose
5-phosphate
PRPP
Phosphoribosyl
amine
Inhibited by AMP
Inhibited by IMP,
AMP, and GMP
AMP
IMP
GMP
Inhibited by GMP
Salvage pathways: re-utilizate purines
O
O P O CH2
OH
OH
O
OH
PPi
+ Base
(Adenine
or Guanine)
PRPP + Adenine
PRPP + Guanine
O
O P O CH2
OH
A-PRT
HG-PRT
OH
A
O
+ PPi
OH
Adenylate/AMP
Guanylate/GMP
There are 2 salvage enzymes with different specificities:
1. Adenine phosphoribosyl transferase (APRT)
2. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Stages of nucleotide metabolism
Endonuclease
Nucleic Acid
Synthesis
Phosphodiesterase
Nucleoside monophosphates
(Mononucleosides)
Nucleoside
triphosphate
Endonuclease
Nucleic Acid
Synthesis
Phosphodiesterase
Nucleotidases
H20
Nucleoside monophosphates
(Mononucleosides)
Pi
Nucleoside
triphosphate
PPi
ADP
Nucleosides
Nucleoside kinase
ATP
Phosphoribosyl
transferases
Pi
PRPP
Phosphorylases
Ribose-1-P
Nucleobases (A, G)
Uric Acid (purines)
Getting Back to ‘Basics’
Nucleotidase
Adenine
Phosphorylase
Adenosine monophosphate
(AMP)
Adenosine
NUCLEOTIDE
NUCLEOSIDE
BASE
Purines in humans are degraded to urate
(ADA)
Gouty Arthritis
The Gout
James Gilray
1799
By Royal Authority
‘King George IV’
George Cruickshank
19th C.
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
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
Gout from increased uric acid production
1. Lost regulation of PRPP Synthase & PRPP Amidotransferase
Ribose
5-phosphate
PRPP
Phosphoribosyl
amine
purines
Inhibited by IMP,
AMP, and GMP
Leads to net increase in biosynthetic/degradation pathways!!
(From slide #18)
Gout from increased uric acid production
2. Defects in salvage pathway lead to increased PRPP & Guanine
PRPP + Guanine
[HG-PRT]
GMP
Leads to net increase in biosynthetic/degradation pathways!!
GOUT Treatment
Hypoxanthine
Guanine
xanthine oxidase
Xanthine
Urate
xanthine oxidase
NaUrate crystals in PMLs
Allopurinol:
a. decrease urate
b. increase xanthine &
hypoxanthine
c. decrease PRPP
Tophi at
helix of ear
1st MTP joint
Initiate urate lowering
therapy !!
Large tophaceous
deposits
surrounding joint
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.
Lesch-Nyhan Syndrome
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
SCID
Severe Combined Immunodeficiency Syndrome
Autosomal recessive disorder
Mutations in ADA*
AMP
H20
Nucleotidase
Pi
Infants subject to bacterial,
candidiasis, viral, protazoal
infections
Adenosine
H20
NH3
Inosine
Adenosine deaminase*
Hypoxanthine
Both T and B cells reduced
(dATP is toxic)
1995-AdV expressing ADA was
successfully employed as gene
therapy strategy
Adenosine Deaminase (ADA) deficiency
Urate
(From slide #19)
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
Summary and Take-Home Points
1. 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
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