Download Purine nucleotide synthesis De novo

Nucleotide Metabolism
(and then, you know…)
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• 1865 – Gregor Mendel: proposes the existence of paired factors of
heredity
• 1869 – Friedrich Miescher: isolates nuclein and discovers that it
was acidic and contained a lot of phosphate
• 1928 – Fred Griffith: observes the transformation of nonpathogenic bacteria through exchange of material with
pathogenic ones
• 1944 – Oswald T. Avery, Colin McLeod and Maclyn McCarty: isolate
the transforming agent -DNA
• 1950 – Erwin Chargaff: the 1:1 ratio of adenine to thymine and
guanine to cytosine
• 1953 – James Watson and Francis Crick (with help from Maurice
Wilkins and Rosalind Franklin): identify the double helical structure
of DNA
• 1977 – Frederick Sanger: DNA sequencing
• 1996 – Ian Wilmut: Dolly the Sheep
• 2003 – The Human Genome Project: the sequencing of 3.2 billion
base pairs along 23 DNA molecules measuring 2 meters
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• 2010 – Craig Venter: synthetic DNA
BIOSYNTHESIS AND DEGRADATION OF NUCLEOTIDES
• Nucleotides are composed of a nitrogenous base, sugar and
phosphate (s)
 base and sugar only = nucleoside
• Nucleotides are precursors of nucleic acids (DNA, RNA),
energy rich compounds (ATP, GTP, UTP, CTP), coenzymes
(NAD/P+ ), FAD, CoA, SAM, Vit B 12) and signaling molecules
(cAMP, cGMP)
• Small amounts of nucleic acids in the diet
• Pancreatic ribonuclease and deoxyribonuclease change the
nucleic acids into their nucleotide monomers
• Intestinal phosphatase converts nucleotides into nucleosides
• The greater portion of nucleosides is modified and excreted
or recycled back to nucleotides
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• The bases of nucleotides are either pyrimidine or purine
• Pyrimidines are characterized by a six-membered heterocyclic
aromatic ring with two nitrogens
• Purines have an additional five-membered imidazole ring
fused with the aromatic ring of pyrimidines
• The common naturally occurring pyrimidines are cytosine,
uracil and thymine (5-methyuracil)
• Purines: cytosine and guanine
• Other naturally occurring purine derivatives include xanthine,
hypoxanthine and uric acid
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• The sugar found in ribonucleotides is D-ribose and in
deoxyribonucleotides is 2-deoxy D-ribose
• The pentose sugars are attached to the nitrogenous bases
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through a β-N-glycosidic bond to give nucleosides
• In pentoses of nucleosides (and nucleotides), the carbon
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numbers are given a prime designation ( ) to distinguish them
from the numbered atoms of the bases
• Nucleosides are named by adding –idine to the root name of
pyrimidines and –osine to the root name of purines: cytidine,
thymidine, uridine, adenosine, guanosine, inosine
• The prefix deoxy- added in the case of deoxyribonucleosides
 Deoxythymidine, deoxyuridine, …
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• Nucleosides are converted to nucleotides through the linking of
a phosphate with the 5’ hydroxyl group of ribose or deoxyribose
 Adenosine 5’-monophosphate (AMP), cytidine 5’
monophosphate (CMP),…
• More phosphates could be added to give nucleoside di- and
triphosphates ; or a single phosphate group can be esterified
with two hydroxyls of the pentose to give a cyclic nucleotide
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• Two types of pathways lead to the synthesis of nucleotides:
the de novo pathways and the salvage pathways
• de novo synthesis begins with amino acids, ribose 5phosphate and CO2
• Salvage pathways recycle free bases and nucleosides
obtained from nucleic acid breakdown (from nucleic acid
turnover or from the diet)
• During de novo synthesis new bases are synthesized
 purine nucleotides are constructed a few atoms at a time
on a ribose-based structure
 the framework for pyrimidine bases is first synthesized
and is then attached to ribose and modified further
• On the other hand, free bases can serve as intermediates in
salvage pathways
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Purine nucleotide synthesis
De novo: is initiated using an activated form of ribose
 Ribose-5-phosphate from PPP reacts with ATP to produce
5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP)
• The pyrophosphate of PRPP is displaced by the amide
nitrogen of glutamine in a reaction catalyzed by glutamine
PRPP amidotransferase
 The committed step of de novo purine synthesis
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• Once 5-phospho-β-D-ribosylamine is formed, the building of the
purine ring begins
 First, the whole of a glycine molecule is added to 5-phosphoβ-D-ribosylamine and phosphoribosylglycinamide is formed
 After eight steps, the first purine nucleotide inosine
monophosphate (IMP) is produced
 Additional nitrogen comes from glutamine and aspartate
 N10-formyl THF and CO2 provide additional carbons
 Four ATP molecules are used in the process
The sources of the atoms in the
purine ring
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• IMP is converted to AMP or GMP:
 the keto group of IMP is replaced by an amino group
(provided by aspartate) in AMP; requires GTP
 the dehydrogenation of IMP using NAD+ yields xanthosine
monophosphate (XMP); XMP is then converted to GMP through
the donation of the amino group of glutamine; requires ATP
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• Nucleoside triphosphates are the most common types of
nucleotide in metabolism
• The production of nucleoside diphosphates requires specific
(for type of base) nucleoside monophosphate kinases whereas
nucleoside triphosphate formation is catalyzed by nucleoside
diphosphate kinases that are not specific for sugar or base
ATP+NMP→ADP+NDP
NTPD+NDPA →NDPD+NTPA
The regulation of de novo purine synthesis
• The committed step is inhibited by excess levels of IMP, GMP
and ATP
• An excess of GMP inhibits the formation of XMP whereas an
excess of AMP inhibits the synthesis of adenylosuccinate
• ATP is needed for the synthesis of GMP and GTP is required
for the synthesis of AMP
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 the pools of the two nucleotides are balanced
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• PRPP synthesis is allostericaly inhibited by ADP, GDP and
other metabolites from other pathways which use PRPP as a
precursor
o Because the de novo pathway consumes much energy, cells
synthesize a significant portion of their nucleotides by salvage
pathways
Purine salvage pathways
• Free purine bases or (deoxy) ribonucleosides can be changed
back to mono nucleotides
• The liver is the major site of purine nucleotide metabolism; it
provides purines and purine nucleosides for other tissues
 in some tissues, the de novo pathway produces little or no
purine nucleotides – these tissues are dependent upon
export from the liver
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• In most tissues AMP is changed by 5’-nucleotidase to adenosine
• Adenosine can then be deaminated by adenosine deaminase
to inosine and inosine can then be converted to IMP
• In adenosine deaminase deficiency large concentrations of
dATP (because adenosine deaminase acts also on
deoxyadenosine) inhibit ribonucleotide reductase;
consequently DNA synthesis is depressed
 Severe combined immunodeficiency (SCID) - T and B
lymphocytes are mainly affected
• Adenosine can also be converted to AMP by adenosine kinase
• Muscular work is accompanied by the production of ammonia,
the immediate source of which is AMP
• AMP is changed to IMP with the associated release of NH4+;
the enzyme involved is known as AMP aminohydrolase or AMP
deaminase
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• IMP is changed back to AMP through the de novo pathway
• The net effect is the generation of fumarate which can serve
an anaplerotic role
 This is known as the purine nucleotide cycle
 It operates in the brain as well as in the muscles
• Adenosine is thought to be a local hormone or autocoid
During strenuous activity, muscles release adenosine
which has a vasodilatory effect leading to increased
delivery of oxygen and nutrients
Adenosine slows the heart rate by blocking the flow of
electrical current
• Purine nucleoside phosphorylase converts inosine, guanosine
and xanthosine to hypoxanthine, guanine and xanthine,
respectively
• Deficiency of purine nucleotide phosphorylase affects T cells
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• Purine bases can react with PRPP under the influence of
phosphoribosyltransferases
• Adenosine phosphoribosyl transferase catalyzes the reaction
of adenine with PRPP to give AMP
• Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
catalyses the reaction of guanine and hypoxanthine with
PRPP
• The deficiency of HGPRT causes the Lesch-Nyhan syndrome
 guanine and hypoxanthine will be degraded instead of
being salvaged
 mental retardation, self-mutilation
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Purine catabolism
• Hypoxanthine is oxidized to xanthine by xanthine oxidase
• Guanine is deaminated by guanine aminohydrolase to xanthine
• Xanthine is converted to uric acid by xanthine oxidase
• Xanthine oxidase is a flavoenzyme with an atom of
molybdenum and four iron-sulfur centers; it produces H2O2
o in some plants xanthine is trimethylated to caffeine
 During periods of extended wakefulness, metabolic
activities in the brain lead to the accumulation of adenosine
 Adenosine induces sleepiness; caffeine promotes
wakefulness by blocking the interaction of extracellular
adenosine with neuronal receptors
• In primates uric acid is excreted in the urine as it is
• In other mammals urate oxidase changes uric acid to allantoin; in
other organisms allantoin to allantoate to urea to NH4+
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• Urate is present in the serum of humans at levels near
precipitation
• This may be because of the use of urate as an anti-oxidant
• Gout (from gutta, “drop”) is a common disorder resulting from
hyperuricemia: > 7 mg/dL in males, > 6 mg/dL in females
• It causes inflammation of the joints– gouty arthritis
 Crystals of sodium urate are deposited in the joints
 The deposition attracts macrophages that engulf the crystals
 Lysosomal membranes of the macrophages are ruptured by
the crystals; this results in the release of lysosomal enzymes
and leukotriene B4 into the tissues
 Histamine is released from mast cells
 Visible structures known as tophi may form near joints
causing deformities
• The deposition of urate crystals within the renal tubules causes
impaired renal function
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• Primary gout results from genetic defects in purine metabolism
 A n overactive PRPP synthase leading to an increase in
purine synthesis
 Deficiency of HGPRT means less salvage reactions
• Glucose-6-phosphatase deficiency leads to the accumulation of
glucose-6-phosphate which will be diverted to ribose-5phosphate and then to PRPP
• Secondary or acquired gout is caused by various factors that can
cause an overproduction of urate or its underexcretion by the
kidneys
• Gout can be treated with dietary control and drugs
• Allopurinol is a suicide inhibitor of xanthine oxidase
 Allopurinol is changed to alloxanthine (oxypurinol) which
is a competitive inhibitor of xanthine oxidase
 Hypoxanthine and xanthine can be excreted in the urine
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• Allopurinol can also react with PRPP producing allopurinol
ribonucleotide thereby reducing purine synthesis
• Colchicine inhibits the polymerization of tubulin into
microtubules
 The division and movement of and phagocytosis by white
blood cells is inhibited
 Lekotriene B4 and histamine release is curtailed
• In earlier times gout was thought to be associated with rich diets
and excessive consumption of alcoholic drinks
• Lead salts used to be added as preservatives in alcoholic drinks;
pots lined with lead-containing materials
• Lead inhibits guanine aminohydrolase leading to the
accumulation of guanine in the joints – Saturnine gout
• The bone stores Pb+2 because it is divalent just like Ca+2; through
time Pb+2 is transported to other tissues including the kidney
• treatment by chelating agents like
ethylenediaminetetraaceticacid (EDTA)
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The de novo biosynthesis of pyrimidine nucleotides
The synthesis of pyrimidines begins with the formation of
carbamoyl phosphate by CPS II – a cytosolic isozyme
Uses glutamine as nitrogen donor instead of free ammonia
Carbamoyl phosphate reacts with aspartate to give carbamoyl
aspartate; the enzyme is aspartate transcarbamoylase
(ATCase)
The closure of the pyrimidine ring is then catalyzed by
dehydration through the action of dihydroorotase
Dihyrdroorotate is then oxidized to orotate by dihydroorotate
dehydrogenase ( a flavoprotein)
 the NADH produced can enter the electron transport chain
CPS II, aspartate transcarbamoylase and dihydroorotate
dehydrogenase are part of a single trifunctional protein
known as CAD
At this point, the basic framework of pyrimidine bases has
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been constructed
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• Orotate and PRPP react under the catalysis of orotate
phosphoribosyl transferase to yield orotidine-5-phosphate
(OMP)
• OMP is then decarboxylated to uridylate (UMP) by OMP
decarboxylase
 UMP is phosphorylated to UTP
 UTP receives an amide group from glutamine to become
cytidine triphosphate (CTP)
• Both orotate phosphoribosyl transferase and OMP
decarboxylase activities are located on a protein called UMP
synthase
 The deficiency of UMP synthase leads to a condition as
orotic aciduria - orotate in the urine, retarded growth,
anemia
 Can be treated with uridine which inhibits the production
of orotate and provides building blocks for nucleic acid
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synthesis
The sources of the atoms in the
pyrimidine ring
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The regulation of de novo pyrimidine synthesis
CPS II is activated by PRPP and inhibited by UTP
As cells approach the S-phase of the cell cycle CPS II becomes
more sensitive to activation by PRPP and less sensitive? to
inhibition by UTP
 phosphorylation of CPS II by MAP kinase at a specific site
leads to an easily activated enzyme while phosphorylation at
another site would lead to an easily inhibited enzyme
The ATCase reaction is the committed step of pyrimidine
synthesis; its regulation in bacteria has been discussed under
allosteric regulation of enzymes
 inhibited by CTP and activated by ATP
The synthesis of thymidylate
DNA contains thymine instead or uracil
The route to thymidine is through deoxyribonucleotides
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• Deoxyribonucelotides are synthesized from the
ribonucleotides by reduction of the 2’-carbon of D-ribose
 The enzyme involved is ribonucleotide reductase
 The substrates are diphosphonucleotides
 The reducing equivalents are ultimately donated by NADPH
via thioredoxin or glutaredoxin
• UDP is changed to dUDP; dUDP is phosphorylated to dUTP
and then pyrophosphate is removed from dUTP to produce
dUMP
• Or dCDP can be changed to dCMP which is deaminated to
dUMP
• dUMP receives a methyl group from N5,N10- methylene THF to
give dTMP which will be phosphorylated to dTTP
 the enzyme is thymidylate synthase
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• The thymidylate synthase reaction converts THF to
dihydrofolate (DHF)
• THF is regenerated through the action of dihydrofolate
reductase with NADPH providing the reducing equivalents
• Rapidly dividing cells require ample supplies of thymidylate for
DNA synthesis; nucleotide biosynthesis can be targeted to
design important drugs
 azaserine is an antibiotic that inhibits glutamine
amidotransferases
 fluorouracil is changed in the body to fluorodeoxyridylate
(FdUMP) which in turn binds to thymidylate synthase
inhibiting it
o Examples of mechanism-based inactivation
 methotrexate (amethopterin) and aminopterin are
competitive inhibitors of dihydrofolate reductase
o Fluorouracil, methotrexate and aminopterin are
important anticancer agents; trimethoprim- is an
antibiotic with a higher affinity for bacterial DHFR 36
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Pyrimidine salvage pathways
• Mammalian cells reutilize a small amount of free pyrimidine
bases
• Pyrimidine phosphorylase can synthesize uridine and
cytidine (through the reaction of uracil and cytosine with
ribose-1-P) but it cannot use thymine
 A ribonucleoside containing thymine is not normally
synthesized in the body
 Instead, thymine will react with deoxyribose-1-P to form
thymidine
• The nucleosides uridine, cytidine, thymidine and
deoxycytidine are phosphorylated to their respective
nucleotides
Thymidine kinase (TK) levels increase significantly
during the S phase of the cell cycle
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Pyrimidine catabolism
• The pyrimidine ring can be degraded unlike that of purines
• Cytosine is degraded through uracil
Cytidine and deoxycytidine are first deaminated to uridine
and deoxyuridine or:
 dCMP (deoxycytidylate) can be deaminated to dUMP
(deoxyuridylate) and 5’-nucleotidase converts dUMP to
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uridine
• Uridine and cytidine are changed to uracil and cytosine by
pyrimidine phosphorylase
• dTMP changed to thymine by the successive actions of 5’nucleotidase and nucleoside phosphorylase
• In addition to CO2 and NH4+ (which enters the urea cycle),
the degradation of uracil gives β - alanine and thymine gives
β- aminoisobutyrate
 β - alanine and β-aminoisobutyrate can be excreted
 Or β - alanine can further be processed to malonyl-CoA
and β-aminoisobutyrate to methylmalonyl-CoA
In some cases β-aminoisobutyrate is produced in
excessive amounts – genetic predisposition (Chinese or
Japanese ancestry) and leukemia (massive cell
destruction)
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