Biosynthesis of Nucleotides 1 - University of Alabama at Birmingham

Report
Synthesis and Degradation of
Nucleotides
Part 1: September 1st, 2009
Champion CS Deivanayagam
Center for Biophysical Sciences and Engineering
University of Alabama at Birmingham
Birmingham, AL 35294-4400
Information Transfer in Cells
The fundamental
process of information
transfer in cells.
Purines and Pyrimidines
Gylocosidic bond
Gylocosidic bond
Note that the numbering are slightly different particularly where the glycosidic bonds are attached
Nucleotide: purines and pyrimides linked to a ribose/de-oxy ribose sugar moiety
Two types of pathways leads to nucleotides:
1. De novo pathway
– Begins with their metabolic precursors: amino acids, ribose-5-phosphate, and CO2
– The free bases A, T, G, C, and U are not intermediates
– Appears to be present in identical form in nearly all living organisms
2. Salvage pathway
– Recycle the free bases and nucleosides released from nucleic acid breakdown
– The free bases are intermediates
I. De Novo Purine Nucleotide Synthesis
The initially synthesized purine derivative is inosine.
Purines are initially formed as ribonucleotides rather than as free bases
AMP
(Adenosine monophosphate)
GMP
(Guanosine monophosphate)
John Buchanan (1948) "traced" the sources of all nine
atoms of the purine ring
Origin of the nine atoms:
Bird feed containing selectively labeled atoms
Examination of the isotope distribution in excreted uric acid (dove
poop research!!!)
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N-1 from aspartic acid
N-3, N-9 from glutamine
C-4, C-5, N-7 from glycine
C-6 from CO2
C-2, C-8 from THF - one carbon units
11 steps lead to the formation
Of IMP (Ionosine mono-phospate)
The names of the enzymes can be a mouthful !!!
In addition each of these enzymes have AKA’s (common name and EC names)
It would be great if you could memorize them, however it is not necessary to memorize
all the steps in this reaction
What you need to learn from this lecture ?
1.
2.
3.
4.
What are the Committed steps that are unique in this synthesis cycle
What are the different feed back inhibition steps in this synthesis cycle
What steps can be utilized to develop inhibitors in this synthesis cycle
What are some of the diseases that are related to this synthesis cycle
A committed step is an effectively irreversible reaction in the biosynthesis pathway
Glutamine PRPP amidotransferase is subject to feedback inhibition
GMP, GDP, GTP as well as AMP, ADP and ATP carry out this action.
How? Through an allosteric site present on the enzyme.
The G series of nucleotides at a Guanine-specific allosteric site on the enzyme
and
The A series of nucleotides at an Adenine-specific allosteric site on the enzyme
Glutamine PRPP amidotransferase is also inhibited by ‘azaserine’
Azaserine acts as an irreversible inhibitor of glutamine-dependent enzymes by covalently
attaching to nucleophilic groups in the glutamine-binding site. It is used as an anti-tumor
agent.
Allosteric enzyme cartoon representation
(aka FGAM synthetase)
(aka Adenylosuccinate lyase)
(aka IMP cyclohydrolase)
In vertebrates these reactions are coupled together
One multifunctional polypeptide chain (110 kda) encodes for:
GAR synthetase (step 3)
GAR tranformylase (step 4)
AIR synthetase (step 6)
Another encodes for
AIR carboxyalse (step 7)
SACAIR synthetase (step 8)
Another polypeptide chain 67 kD (organized as 135 kDa dimers)
AICAR tranformylase (step 10)
IMP synthase (step 11)
Folate Analogs as Antimicrobial and Anticancer Agents
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De novo purine biosynthesis depends
on folic acid compounds at steps 4 and
10
For this reason, antagonists of folic
acid metabolism indirectly inhibit
purine formation and, in turn,
nucleic acid synthesis, cell growth,
and cell development
Rapidly growing cells, such as infective
bacteria and fast-growing tumors, are
more susceptible to such agents
Sulfonamides are effective antibacterial agents
Methotrexate and aminopterin are folic
acid analogs that have been used in
cancer chemotherapy
Step 12: Synthesis of Adenine and Guanine Ribonucleotides:
a). AMP is made from IMP in two steps.
The first step converts IMP to
adenylosuccinate.
The second step is
catalyzed by adenylosuccinate lyase that
produces AMP.
b). The formation of GMP from IMP
requires oxidation at C-2 of the purine ring,
followed by a glutamine-dependent
amidotransferase reaction that replaces the
oxygen on C-2 with an amino group to yield
2-amino,
6-oxy
purine
nucleoside
monophosphate – i.e., GMP. The second
reaction is catalyzed by GMP synthetase,
shown here.
The Purine Biosynthetic Pathway is Regulated at Several Steps
Allosteric regulation occurs in the first two steps, and AMP and GMP are competitive
inhibitors in the two branches at right.
Can Cells Salvage Purines?
•
Nucleic acid turnover (synthesis and degradation) is an ongoing process in most cells
•
Purines that are obtained from diet and turn over and not degraded can be reconverted to
nucleosided tri-phosphates and be reused.
•
Nucleotides are then converted to nucleosides by specific nucleotidases and non-specific
phosphotases
NMP + H2O  nucleoside + Pi
•
Nucleosides are hydrolyzed by nucleosidases or nucleoside phophorylases to release the
purine base
Nucleoside + H2O Nucleosideases base + ribose
Nucleoside + Pi  Nucleoside phosphorylase  base + ribose-1-P
•
Salvage pathways collect hypoxanthine and guanine and recombine them with PRPP to
form nucleotides in the HGPRT reaction
Major pathways of purine catabolism in animals.
Major pathways of purine
catabolism in animals.
Animals Oxidize Uric Acid to Different Excretory Products
HGPRT Converts Bases Back to Nucleotides
Notice: PRPP is also involved in the
salvage pathway.
HGPRT - Hypoxanthine-guanine phosphoribosyltransferase
Lesch-Nyhan Syndrome – HGPRT Deficiency Leads to a Severe Disorder
Absence of HGPRT is the
cause of Lesch-Nyhan
syndrome
In L-N, purine synthesis is
increased 200-fold and
uric acid is elevated in
blood
Victims of Lesch-Nyhan
syndrome experience severe
arthritis due to accumulation
of uric acid, as well as
retardation, and other
neurological symptoms.
Lesch-Nyhan syndrome
results from a complete
deficiency in HGPRT.
Major pathways of purine catabolism in animals.
Lack of Adenosine Deaminase is One Cause of Severe Combined Immunodeficiency
Syndrome
SCID is a group of related disorders involving diminished immune responses. 30% of
SCID patients lack the enzyme adenosine deaminase. In the absence of ADA,
deoxyadenosine is not deaminated to deoxyinosine as normal (above).
Gout is a Disease Caused by an Excess of Uric Acid
• Xanthine oxidase (XO) in liver, intestines (and milk) can oxidize hypoxanthine
(twice) to uric acid
• Humans and other primates excrete uric acid in the urine, but most N goes out
as urea
• Birds, reptiles and insects excrete uric acid and for them it is the major
nitrogen excretory compound
• Gout occurs from accumulation of uric acid crystals in the extremities
• Precipitation and deposition of uric acid causes arthritic pain and kidney
stones
• Causes: impaired excretion of uric acid and deficiencies in HGPRT
Allopurinol, an analog of hypoxanthine, is a potent inhibitor
of xanthine oxidase.
Allopurinol binds tightly to xanthine oxidase, preventing uric
acid formation. Hypoxanthine and xanthine do not
accumulate to harmful concentrations because they are
more soluble and thus more easily excreted.
Summary of 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
Reading assignment:
glucose 6-phosphatase
Hyperuricemia
Tomorrow:
1. Pyrimindine synthesis/degradation
2. Deoxyribonucleotide synthesis

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