Transcript 5-1 Necleotide Metabolism

FIFTH WEEK
Gihan E-H Gawish, MSc, PhD
Ass. Professor
Molecular Genetics and Clinical Biochemistry
KSU
Purine metabolism (Overview)
1. Nomenclature/nucleotide structure
2. Extracellular Hydrolysis of Ingested Nucleic Acid
3. De novo synthesis pathways
4. Re-utilization pathways
5. Metabolic diseases of purine Metabolism (Gout,
Lesch-Nyhan, SCID)
DNA
RNA
1- Endonucleases
2-Phosphodiesterase
Oligonucleotides
3- Phosphorylase
Ribose + Free Bases
Nucleotides
Nucleic Acid met abolism
Click on any blue rect angle t o see det ails.
Carbamoyl
Phosphate
am i n o a c id s,
f o la t e
Purine
Salvage
PRPP
Purine
Biosynt hesis
IMP
Pyrimidine
Biosynt hesis
OMP
Pyrimidine
Salvage
Pyr im idine MP
Pur ine MP
Ribonucleot ide
reduct ase
Ribonucleot ide
reduct ase
dNTP
NTP
Purine
Degradat ion
DNA
RNA
dNTP
NTP
Pyrimidine
Degradat ion
NH4
Uric A cid
( energ y )
The nomenclature of purines depends
on their linkage to a pentose
Adenine
Base
Adenosine
Nucleoside*
Base
Adenosine Monophosphate
Nucleotide
Base (P04 ester)
* when the base is purine, then the nucleoside ends in OSINE (AdenOSINE, GuanOSINE, InOSINE)
The active forms of nucleotides in biosynthesis
and energy conversions are di-and tri-phosphates
(i)
(ii)
Nucleoside Monophosphate Kinase
CMP + ATP
CDP + ADP
Nucleoside Diphosphate Kinase
XDP + YTP
XTP + YDP
Structures of Common Purine Bases.
H= 6 oxy purine
X= 2,6 dioxy purine
A= 6 amino purine
G= 2 amino, 6-oxy purine
Hypoxanthine is an intermediate for Adenine and Guanine
(N source)
Aspartate
(N source)
Glutamine
The common mechanistic them for the conversion of A and G is the conversion of
a carbonyl oxygen to an amino group
There are two basic mechanisms to
generate purines and pyrimidines
1. DE NOVO BIOSYNTHETIC PATHWAYS
(building the bases from simple building
blocks)
2. SALVAGE PATHWAYS
(the reutilization of bases from dietary
or catabolic sources)
The biosynthesis of purine (A and G) begins with the
synthesis of the ribose-phosphate
Pentose phosphate
pathway
Ribose phosphate
pyrophospho-KINASE
The major regulatory step in purine biosynthesis is the conversion
of PRPP to 5-Phosphoribosyl-1-amine
*
Glutamine
PRPP
Glutamate
PPi
Amidophosphoribosyl
transferase
Amidophosphoribosyl transferase is an important regulatory enzyme in purine
biosynthesis. It is strongly inhibited by the end products IMP, AMP, and GMP.
This type of inhibition is called FEEDBACK INHIBITION.
Several amino acids are utilized in purine biosynthesis
IMP is the precursor for both AMP and GMP, the base is also called
hypoxanthine
Conversion of Hypoxanthine to Adenine/Guanine.
(N source)
Aspartate
(N source)
Glutamine
The common mechanistic theme for the conversion of A and G is the
conversion of a carbonyl oxygen to an amino group
Purines: where do the atoms come from?
Purine intermediates include:
1. Glycine
2. 1 C units of 5,10 mTHF
3. Glutamine
4. Asparate
The regulation of purine biosynthesis is a
classic example of negative feedback
Inhibited by AMP
AMP
Ribose
5-phosphate
PRPP
Phosphoribosyl
amine
IMP
GMP
Inhibited by IMP,
AMP, and GMP
Inhibited by GMP
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
Uric Acid (purines)
Salvage pathways for the re-utilization of purines;
There are 2 salvage enzymes with different specificity;
1. Adenine phosphoribosyl transferase
2. Hypoxanthine-guanine phosphoribosyl transferase
O
O
O
P O CH2
OH
OH
O
PPi
+
Base
(ie Adenine)
O
OH
P O CH2
OH
OH
A
O
OH
A-PRT
PRPP + Adenine
Adenylate
HG-PRT
PRPP + Guanine
Guanylate
+
PPi
Purines in humans are degraded to Urate
Important points:
1. Nucleotides are constantly
undergoing turnover!
2. There are many enzymes
involved;
Nucleotidases
Nucleoside phosphorylases
Deaminases
Xanthine oxidases
3. the final common intermediate
in
humans is Urate, which is
excreted.
4. there are several metabolic
disorders
resulting from defects in purine
catabolism.
NH 2
HO
2
+
4
N
N
N
N
ribose
O
NH
N
HN
Adenosine
deaminase
(ADA)
ribose
purine nucleoside
phosphorylase
N
N
H
Hypoxanthine
Inosine
Adenosine
N
HN
N
N
O
ribose - 1 - P
Pi
xanthine
oxidase
Pi
O
N
HN
NH 2
ribose - 1 - P
N
N
ribose
O
Guanosine
N
N
H
O
+
NH 4
N
HN
purine nucleoside NH
2
phosphorylase
H 2O
N
HN
Guanine
deaminase
O
Xanthine
Guanine
xanthine
oxidase
O
Catabolism of Purine Nucleotides
N
H
N
H
HN
O
Uric acid
O
H
N
N
H
N
H
Disorders of Purine Metabolism:
Disorder
Gout
Lesch Nyhan
syndrome
Defect
PRPP synthase/
HGPRT
lack of HGPRT
Comments
Hyperuricemia
Hyperuricemia
SCID
ADA
High levels of dAMP
von Gierke’s disease
glucose -6-PTPase
Hyperuricemia

a disorder associated with abnormal amounts
of urates in the body

early stage: recurring acute non-articular
arthritis

late stage: chronic deforming polyarthritis
and eventual renal complication

disease with rich history dating back to
ancient Greece
What happens in gout?
Inhibited by AMP
AMP
Ribose
5-phosphate
PRPP
Phosphoribosyl
amine
IMP
GMP
Inhibited by IMP,
AMP, and GMP
Inhibited by GMP
1. Negative regulation of PRPP Synthatase & PRPP Amidotransferase is lost
2. PRPP levels are increased because of defects in salvage pathways
Therefore, there is net increase in biosynthetic/degradation pathways!!
GOUT (Gouty Arthritis): A defect of purine metabolism
Serum Uric Acid Levels
(mg/dl)
Incidence of Gout
(% of cases)
>9.0
7-9
<7.0
~10%
0.5-3.5%
0.1%
Hypoxanthine
Guanine
xanthine oxidase
Xanthine
Urate
xanthine oxidase
Allopurinol:
a. decrease urate
b. increase xanthine &
hypoxanthine
c. decrease PRPP

prevails mainly in adult males

rarely encountered in premenopausal women

symptoms are cause by deposition of crystals
of monosodium urate monohydrate (can be
seen under polarized light)

usually affect joints in the lower extremities
(the big toe is the classic site)
SCID-Severe Combined Immunodeficiency Syndrome
Autosomal recessive disorder
Mutations in ADA
AMP
H20
Nucleotidase
Pi
Both T and B cells are significantly
reduced (dATP is toxic)
Adenosine
H20
Adenine deaminase*
NH3
Inosine
Infants subject to bacterial,
candidiasis, viral, protazoal
infections
Hypoxanthine
1995-AdV expressing ADA was
successfully employed as gene
therapy strategy
Degradation
of AMP
OH
NH2
NH3
H2 O
N
N
N
N
AMP deaminase
N
N
N
N
Ribose-P
Ribose-P
H2 O
H2O
Nucleotidase
Nucleotidase
Pi
Pi
O
NH2
NH3
H2 O
N
N
HN
N
Adenosine deaminase
N
N
N
Pi
N
Ribose
Ribose-1-P
Ribose
Purine nucleoside
phosphorylase
O
may be reused
through
salvage pathway
N
HN
N
N
H
hypoxanthine
NH2
OH
H2N
N
N
N
HN
N
N
N
N
Ribose
Ribose
transition state
H
OH
N
HN
N
N
O
Ribose
Pentostatin
(a transition state analog)
shows promise in combination
with Ara-C (Vidarabine)
N
HN
N
N
Ribose
MECHANISM OF ADENOSINE DEAMINASE
In the absence of ADA lymphocytes are destroyed
deoxyadenosine is not destroyed, is converted to
dAMP and then into dATP
 dATP is a potent feedback inhibitor of
deoxynucleotide biosynthesis
 this leads to SCID (severe combined
immunodeficiency disease)
 Infants with this deficiency have a high fatality rate
due to infections





treatment consists of administering
pegylated ADA which can remain in the blood
for 1 – 2 weeks
more efficient is gene therapy: replacing the
gene that is missing or defective
gene therapy has been performed on
selected patients
Degradation of GMP and XMP
O
O
H
N
N
H2N
N
H
N
O
N
N
Ribose-P
N
N
H
Ribose-P
H2O
H2O
nucleotidase
nucleotidase
Pi
Pi
O
O
H
N
N
N
N
N
N
H2N
H
O
Pi
N
N
Ribose
PNP
Ribose
H
Ribose-1P
Pi
PNP
Ribose-1P
O
O
H
N
N
H
N
N
H2N
N
N
H
H2O
NH3
O
N
N
H
H
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