Transcript Purine metabolism - mustafaaltinisik.org.uk

Nucleotides play key roles in many, many cellular processes
1. Activated precursors of RNA and DNA
2. Adenine nucleotides are components of three major
co-enzymes, NAD, FAD, and CoA
3. Nucleotide derivatives are activated intermediates in
biosynthetic processes (UDP-glucose, SAM)
4. Serve as metabolic regulators (for example cAMP and
the activation of cell signaling.
5. Serve as major currency of energy in all cells
(ATP and GTP).
6. Many metabolic diseases have their etiology in nucleotide
metabolism.
Purine metabolism (Overview)
1. Nomenclature/nucleotide structure
2. De novo synthesis pathways
3. Re-utilization pathways
4. Metabolic diseases of purine
Metabolism(Gout, Lesch-Nyham,
SCID)
Why go through to the trouble to convert Uracil to Thymine?
dUMP
Thymidylate synthase
reduced
N5,N10-methylenetetrahydrofolate
dTMP
oxidized
Dihydrofolate
NADPH
Serine
transhydroxymethylase
Dihydrofolate
reductase
Tetrahydrofolate
NADP+
Thymidylate
Synthase
The nomenclature of purines and pyrimidines depends on their linkage to a pentose
Cytosine
Base
Cytidine
Nucleoside*
Base
Cytidine Monophosphate
Nucleotide
Base (P04 ester)
* when the base is purine, then the nucleoside ends in OSINE (AdenOSINE, GuanOSINE, InOSINE)
when the base is pyrimidine, then the nucleoside ends in IDINE (UrIDINE, CytIDINE, ThymIDINE)
The active forms of nucleotides in biosynthesis
and energy conversions are di and triphosphates
Nucleoside Monophosphate Kinase
CMP + ATP
CDP + ADP
Nucleoside Diphosphate Kinase
XDP + YTP
XTP + YDP
RIBONUCLEOTIDE REDUCTASE
1. Complex enzymatic reaction whereby electrons
are transferred from NADPH through a series of
sufhydryl groups at the catalytic site of Ribonucleotide
Reductase.
NADPH
NADP+
2. Active site of RR contains thioredoxin, a 12 kD protein
with two exposed cysteines, which become oxidized.
3. This ultimately allows for the reduction of ribose.
REGULATION
1. Based on the response to cellular need for dATPs.
dATP is general inhibitor
ATP is a general activator
Nucleotides are linked by 5’ to 3’ phosphodiester bonds to generate DNA and RNA
Structures of Common Purine Bases.
H= 6 oxy purine
X= 2,6 dioxy purine
A= 6 amino purine
G= 2 amino, 6-oxy purine
Structures of Common Purine Bases.
H= 6 oxy purine
X= 2,6 dioxy purine
A= 6 amino purine
G= 2 amino, 6-oxy purine
Structures of Common Purine Bases.
(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 pyrophosphoKINASE
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 a 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
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Structures of Common Purine Bases.
(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
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
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Nucleotidase
Phosphorylase
Cytosine
Base
Cytidine
Nucleoside*
Base
Cytidine Monophosphate
Nucleotide
Base (P04 ester)
Salvage pathways for the re-utilization of purines;
There are 2 salvage enzymes with different specificities;
1. Adenine phosphoribosyl transferase
2. Hypoxanthine-guanine phosphoribosyl transferase
QuickTime™ and a
+ PPi
decompressor
+ GIF
Guanine
are needed to see this picture.
A-PRT
PRPP + Adenine
Adenylate
HG-PRT
PRPP + Guanine
Guanylate
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!!
“The Gout” James Gilray, 1799.
“By Royal Authority”
by George Cruickshank.
19th century.
David Wells
New York Yankees
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.
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
SCID-Severe Combined Immunodeficiency Syndrome
Autosomal recessive disorder
Mutations in ADA
AMP
H20
Nucleotidase
Pi
Adenosine
Both T and B cells are significantly
Reduced (dATP is toxic)
H20
Adenine deaminase*
NH3
Inosine
Infants subject to bacterial,
candidiasis, viral, protazoal
infections
Hypoxanthine
1995-AdV expressing ADA was
sucessfullly employed as gene
therapy strategy
Disorders of Purine Metabolism:
Disorder
Gout
Lesch Nyhan
syndrome
Defect
PRPP synthase/
HGPRT
lack of HGPRT
SCID
ADA
von Gierke’s disease
glucose -6-PTPase
Comments
Hyperuricemia
Hyperuricemia
high levels of dAMP
Hyperuricemia
Structure of Pyrimidines
C= 2 oxy, 4 amino pyrimidine
T= 2,4 dioxy 5-methyl pyrmidine
U= 2,4 dioxy pyrimidine
O= 2,4 dioxy 6 carboxy pyrimidine
Pyrimidines: where do the atoms come from?
Pyrimidine biosynthesis:
(occurs in cytosol)
Pyrimidine biosynthesis begins with the assembly of the ring, then linked
To ribose phosphate.
Precursors are Glutamine (NH2), Bicarbonate (C) , and ATP (PO4).
Q. Why is it advantageous to generate carbamoyl phosphate in the cytosol
rather than the mitochondria?
ATCase is the committed step in pyrimidine biosynthesis
committed step in
pyrimidine biosynthesis
Carbamoyl phosphate synthase II, ATCase, and Dihydrooratase are
linked in a single 240 kD polypeptide chain. The enzyme is sometimes
referred to as CAD.
The second phase of pyrimidine biosynthesis
QuickTime™ and a
GIF decompressor
are needed to see this picture.
in pyrimidine biosynthesis, the addition of ribose phosphate
*Note,
moiety occurs late in the pathway, via its addition of Orotate.
*
QuickTime™ and a
GIF decompressor
are needed to see this picture.
QuickTime™ and a
GIF decompressor
are needed to see this picture.
ATCase is feedback inhibited by the end-products of
pyrimidine biosynthesis
C02 + Glutamine + ATP
Rate
Carbonyl Phosphate
ATP
CTP
Inhibited by CTP
[Aspartate]
Carbonyl Asparate
UMP
UTP
CTP
Why go through to the trouble to convert Uracil to Thymine?
dUMP
Thymidylate synthase
reduced
N5,N10-methylenetetrahydrofolate
dTMP
oxidized
Dihydrofolate
NADPH
Serine
transhydroxymethylase
Dihydrofolate
reductase
Tetrahydrofolate
NADP+
Thymidylate
Synthase
QuickTime™ and a
GIF decompressor
are needed to see this picture.
Common chemotherapeutic drugs act
at the level of dTMP synthesis.
Fluorodeoxyuridylate (5-FU)
dUMP
Thymidylate synthase
reduced
N5,N10-methylenetetrahydrofolate
dTMP
oxidized
Dihydrofolate
NADPH
Dihydrofolate
reductase
Tetrahydrofolate
NADP+
Methotrexate
AZT is used to inhibit HIV reverse transcriptase (RNA-dependent DNA pol)
N=N=N
3’ azido-2’3’ dideoxythymine (AZT)
This class of compounds (chemotherapeutics, viral inhibitors, etc are called
nucleoside analogs.
Common side effects of DNA inhibitor chemotherapeutics:
Diarrhea
Skin and eye sensitivity to sunlight
Abnormal liver function tests
Hair loss
Immuno-suppression
Skin rashes
Fatigue
Headache, backache,
Spinal cord irritation
Peripheral neuropathies
Summary:
1. Recognize basic structures of purines and pyrimidines
2. Key regulatory enzymes and feedback networks
3. Targets for clinical interventions