Transcript Guanine Nucleotide

SFA 2073
NUCLEOTIDES:
STRUCTURE & METABOLISM
Nik Norma Nik Mahmood (Ph.D)
U.N.S.W Sydney
Objectives
At the end of the lecture, you should be able to:
1- List the precursors of purine synthesis.
2- Describe purine de novo synthesis & its regulation.
3-Describe salvage synthesis of purine.
4-Describe purine catabolism & urate formation.
5-Explain biochemical basis of diseases of purine
metabolism defects such as gout.
6-Explain the action of Allopurinol in treatment of gout.
Discussion flow
• Nucleotide?
- structure  components
- types
• Nucleotide pool:
- incomming by (i) de novo pathways
(ii) salvage pathways
- outgoing : conversion to other form or breakdown to
smaller end products
NUCLEOTIDES - introduction
• A nucleotide  a molecule comprises of phosphoric acid,
sugar ribose (in RNA) or deoxyribose (in DNA), and an
organic base (derivative of purines or pyrimidines).
• There are 2 types ; deoxyribose nucleotides (in DNA ) and
ribose nucleotides ( in RNA).
• Nucleotides are phosphorylated nucleosides.
In the nucleoside, the
base is bonded through
a β-N-glycosidic bond
to the anomeric carbon
(C1)of the
ribose/deoxyribose and
N9 of purine or N1 of
pyrimidine. The base is
in the anti-orientation*)
•
Skeleton
derivatives
purine:
adenine
guanine
pyrimidine
uracil
thymine
cytocine
 deoxyribonucleosides
• Beside the sugar unit, the base component in DNA and
RNA differ slightly:
DNA: adenine, guanine, cytosine and thymine
RNA: adenine, guanine, cytosine and uracil
Nucleotide ( XMP; XDP; XTP):- X= nucleoside ;M= mono,
D= di, T= tri, P=phosphate
Adenine : (RNA) AMP, ADP, ATP; (DNA ) dAMP, dADP,
dATP
Guanine : (RNA) GMP, GDP, GTP; (DNA ) dGMP, dGDP,
dGTP
Cytosine : (RNA) CMP, CDP, CTP; (DNA ) dCMP, dCDP,
dCTP
Thymine : (DNA ) dTMP, dTDP, dTTP
Uracil : (RNA) UMP, UDP, UTP
Naming : CMP: cytosine mono phosphate ,
GDP: guanosine di phosphate; ATP: adenosine tri
phosphate
• A nucleotide can be mono, di or tri phosphorylated.
• The first phosphate group is bonded to the 5'-carbon of the sugar
unit.
3rd
1st
2nd
deoxyguanosine Triphosphate (GTP)
Adenosine Triphosphate (ATP)
Cytosine Triphosphate (CTP)
Guanosine Triphosphate (GTP)
Uridine Triphosphate (UTP)
syn-Adenosine
anti-Adenosine
Orientation of sugar/base in adenosine
• Two nucleotides are condensed by the reaction
between the alcohol of a 5'-phosphate of one
nucleotide and the 3'-hydroxyl of a second, with the
elimination of H2O, forming a phosphodiester bond.
• NUCLEOTIDE also are required for numerous other
important functions within the cell. These functions
include
1. serving as energy stores for future use in phosphate
transfer reactions. These reactions are predominantly
carried out by ATP.
2. forming a portion of several important coenzymes
such as NAD+, NADP+, FAD and coenzyme A.
3. serving as mediators of numerous important cellular
processes such as second messengers in signal
transduction events. The predominant second
messenger is cyclic-AMP (cAMP), a cyclic derivative of
AMP formed from ATP.
4. controlling numerous enzymatic reactions through
allosteric effects on enzyme activity.
5. serving as activated intermediates in numerous
biosynthetic reactions. Eg of activated
intermediates (i) S-adenosylmethionine (S-AdoMet)
involved in methyl transfer reactions (ii) sugar
coupled nucleotides involved in glycogen and
glycoprotein synthesis
6. precursors to DNA & RNA synthesis
•
Purine bases and purine nucleosides are toxic to
humans so must be readily eliminated.
• Purine and pyrimidine nucleotides, is metabolized
via its specific pathway
NUCLEOTIDE METABOLISM
• Discusses pathways that lead to:
i - breakdown of nucleotides
ii - [ ] of nucleotides
▪ de novo pathway i.e synthesis from precursor
▪ salvage pathway ( recycling)
Salvage
(recycling)
DNA/RNA synthesis
Nucleotides Pool
Biosynthesis
from precursor
degradation
• Most, but not all, nucleic acids in cell (animal or plant) are
associated with protein═> nucleoprotein eg chromatin,
ribosomes, viruses.
• Dietary nucleoprotein is split by pancreatic enzymes (in
stomach) and tissue nucleoprotein by lysosomal enzymes.
• After dissociation, the protein is metabolized like any other
protein.
• The nucleic acids are hydrolyzed randomly by nucleases to
yield a mixture of shorter polynucleotides.
Cellular nucleoprotein
Lysosomal enz
Protein + N.A nucleases
Pacreatic enz
Diet nucleoprotein
Poly
nucleotides
Endo & exo
Nucleases, and
phosphodiesterases
Triphospho,
monophosphon’tide,
nucleotidase
deaminase
Salvage
(recycling)
free
bases
• shorter polynucleotides are hydrolyzed by endo &
exonucleases(endo & exonucleotidase) to yield tri and
mononucleotides (nucleotidase = diesterase )
• The triP cleaved by phosphodiesterases to the
mononucleotides: AMP, GMP, CMP, UMP and TMP
• The pathway of purine and pyrimidine n’tide differ after
this point
• The mononucleotides are hydrolyzed by nucleotidases
and purine/pyrimidine nucleoside phosphorylase to free
base.
• The free base undergoes either catabolic pathway or
salvage pathway.
 release of free base from N.A occurs in 2 stages:
i- hydrolysis of phosphoester bonds in N.A
nucleotides
ii- hydrolysis of phosphoester and glycosidic bonds in
nucleotide  free base
Catabolism/Degradation of purine and
pyrimudine (fate of free base) - IN LIVER.
free bases
end products
• breakdown of base structure
• conversion to 5’ mononucleotide – Salvage
pathway
I. Catabolism(Degradation) of the Bases.
Purine and paramidine are metabolized differently:
• End product of purine base catabolism/degradation is
uric acid.
• End product of pyrimidine base catabolism
(degradation) is β- alanine and β- amino Isobutyrate
II. Characteristic of catabolism of purine base :
- Pathway for adenine differs from that for guanine.
- Pathway lead to similar final product that is Uric Acid.
- No base ring-cleavage
Catabolism of Purine Nucleotides
• Groups attached to the purine ring are sequentially
removed from AMP & GMP by parallel pathways:
1- Phosphate groups are removed by nucleotidase.
2- Amino groups are released by:adenosine deaminase &
guanine deaminase (or guanese).
3-The pentoses are removed by purine nucleoside
phosphorylase .
• The sum of these reaction converts AMP & GMP to
hypoxanthine & xanthine respectively.
Guanine Nucleotide (GMP)
i)
GMP is acted upon by nucleotidase producing
guanosine and Pi.
ii) Guanosine is further acted upon by purine
nucleoside phosphorylase liberating free
guanine + dribose
iii)Guanese acts upon guanine to create Xanthine.
iv) Xanthine oxidase acts upon xanthine to create
Uric acid. This enzyme is clinically important
Adenine Nucleotide (AMP)
• Degradation occurs by either
(i) AMP is acted upon by nucleotidase liberating
adenosine which is further acted by adenosine
deaminase producing inosine
OR
(ii) AMP is acted upon by AMP deaminase producing
inosine monophosphate (IMP)
- IMP is then acted upon by nucleotidase liberating
inosine
INOSINE is further acted to form final product xanthine:
AMP
AMP deaminase
inosine
phosphorylase
IMP
nucleotidase
hypoxanthine
inosine
Xanthine oxidase
xanthine
nucleotidase
Pi
ribose
guanese
excrete
Catabolism of Guanine Nucleotide (GMP) & Adenosine Nucleotide AMP
• Xanthine is acted upon by Xanthine oxidase to form urate.
• Urate is transported to and excreted by the kidney into the
urine.
• Urate is not very soluble but is not a problem to kidney for
excretion.
• When the urine is very acid or has high [Ca2+]; [Na+], urate
salts co precipitate with calcium or sodium salts and can
form stones in kidney or bladder. A very high concentration
of urate in the blood leads to a fairly common group of
diseases referred to as gout (intense pain with swelling).
• To reduce [plasma urate], is to reduce urate synthesis which
is catalysed by Xanthine oxidase. This is key enzyme. Its
activity is inhibited by drug ‘allopurinol’ which is structurally
similar to xanthine
• In birds, uric acid is further degraded to a high water
soluble end product, allantoin
Summary
Guanine
• A nuclease frees the nucleotide
• A nucleotidase creates guanosine
• Purine nucleoside phosphorylase converts guanosine to
guanine
• Guanase converts guanine to xanthine
• Xanthine oxidase converts xanthine to uric acid (urate)
Adenine
• A nuclease frees the nucleotide
– A nucleotidase creates adenosine, then adenosine
deaminase creates inosine
– Alternatively, AMP deaminase creates inosinic acid, then a
nucleotidase creates inosine
• Purine nucleoside phosphorylase acts upon inosine to create
hypoxanthine
• Xanthine oxidoreductase acts upon hypoxanthine to create
xanthine
• Xanthine oxidoreductase acts upon xanthine to create uric
acid
Clinical Significances of Purine Metabolism
• Clinical problems result of abnormal catabolism of
purine is due to insolubility of uric acid.
Gout (hyperuricemia)
• Excess accumulation of uric acid .
• GOUT results from the precipitation of sodium urate
crystals (tophi) in the synovial fluid of joints, leading to
severe inflammation,arthritis & severe degeneration of
joints.
• Often attacks first metatarsophalangeal joint of big
toe.
• Gout is treated by allopurinol.
• Allopurinol is a structural analog
of hypoxanthine that strongly inhibits xanthine
oxidase.
Uric acid normal limits are
4-7mg/dl for males &
3 - 6mg/dl for females.
Severe gout in the fingers
resulting in large, hard
deposits of crystals of uric
acid. These deposits are called
tophi.
Causes of hyperuricemia
I- Overproduction of purine due to:
1-Specific enzyme defects:
A- Increased activity of PRPP synthase.
B- PRPP amidotransferase is less sensitive to the feedback
inhibition by purine nucleotides.
C-Deficiency of salvage enzymes ( HGPRT), so
consumption of PRPP is decreased leading to its
accumulation as in Lesch Nyhan syndrome.
D-Deficiency of glucose-6-phosphatase & enhanced
conversion of g-6-p to ribose 5- phosphate and PRPP as in
Von Gierke’s disease
2- Secondary to other diseases e.g. cancer that enhance
tissue turnover and overload of purines.
II- Defective elimination of urate ( renal disorder)
III- Genetic defect
1. Lesch-Nyhan syndrome
• Due to loss of a functional HGPRT gene, so consumption of PRPP
is decreased leading to uric acid accumulation.
• Patients exhibit severe gout & severe malfunction of the nervous
system, mental retardation, spasticity & self harm (selfmutilation).
• Death usually occurs before the age of 20 year.
2. Hypouricemia
• Severe combined immunodeficiency disease (SCID)
• SCID is a group of inherited disorders characterized by the lack
of immune response to infectious diseases.
• This is due to the inability of B &T lymphocytes to proliferate &
produce antibodies.
• SCID patients suffer from a deficiency in the enzyme adenosine
deaminase (ADA) (~30%).
• In the absence of ADA, deoxyadenosine is not degraded and
converted into dAMP and then into dATP.
continuous
•dATP is a potent feedback inhibitor of deoxynucleotide
biosynthesis. So, DNA synthesis is impaired.
•Rapidly proliferating lymphocytes are particularly
susceptible if DNA synthesis is impaired, & seriously
impairs the immune responses.
• The disease is usually fatal in infancy .
•A less severe immunodeficiency results when there is a
lack of purine nucleoside phosphorylase (PNP)
(ribonucleotide reductase & DNA synthesis are inhibited
due to accumulation of dGTP).
Catabolism/Degradation of pyrimidine
nucleotides
pyrimidine
nucleotides
nucleotidases
pyrimidine nucleoside
pyrimidine nucleoside phosphorylase
pyrimidine
• The pyrimidine are then degraded further into βalanine and β- amino isobutyrate involving ring
cleavage:
- Atoms 2 and 3 of both rings are released as ammonia
and carbon dioxide.
-The rest of the ring is left as a beta-amino acid.
• Beta-amino isobutyrate from thymine or 5-methyl
cytosine is largely excreted. Beta-alanine from
cytosine or uracil may either be excreted or
incorporated into the brain and muscle dipeptides,
carnosine (his-beta-ala) or anserine (methyl hisbeta-ala).
Salvage pathway:
• Is a metabolic pathway that uses substrates other
than the usual biosynthetic intermediates for a
product, eg. free purines from the hydrolysis of
nucleotides (from diet & intracellular N.A) is
salvaged for the generation of new nucleotides
• PRPP is the donor of phosphorylribose to the base. The
reaction is catalyzed by phosphoribosyl transferase
enzyme.
• It requires far less energy than de novo synthesis.
• Mammalian liver provides purine bases & nucleosides
for salvage to tissues incapable for their biosynthesis
e.g- brain cells, RBCs, & WBCs
Mechanisms of salvage pathway
1- Phosphoribosylation of purines:
A- Hypoxanthine-Guanine Phosphoribosyl transferase
[HGPRT].
• This enzyme transfers ribose 5- phosphate from PRPP to
the purine ring (hypoxanthine & guanine) resulting in IMP
& GMP respectively.
PRPP
Hypoxanthine
HGPRT
PPi
IMP
continue
PPi
PRPP
GMP
Guanine
HGPRT
B- Adenine Phosphoribosyl transferase [APRT].
PRPP
Adenine
PPi
AMP
APRT
2- Direct phosphorylation of purine nucleosides:
ATP
AMP
ADP
Adenosine kinase
ADP
Anabolism of base
• Synthesis of purine bases from precursors
- The starting step is formation of PRPP from ribose 5’phosphate & ATP
- the final form of purines in its synthesis process is as
the ribonucleotides. The synthesis starts of with
5-Phosphoribosyl-1-pyrophosphate (PRPP) which is
the activated form of ribose 5-phosphate.
- occurs in the cytosol of the liver cells.
Origin of atoms in the purine
─ This rxn occurs in many tissue types..
– sensitive to di- and tri-phosphates, and 2,3-DPG
• replacement of the pyrophosphate of PRPP by the amide group of
glutamine, 5-phosphoribosylamine is formed. This rxn is catalysed by
glutamine PRPP amidotranferase (a dimer ) and is the rate
determining step (control/regulated point)
RDS
Regulation of
• A series of additions take place to make first the
5- and then the 6-membered ring.
• The whole glycine molecule, adds to the amino
group to be atoms 4, 5, and 7 of the purine ring.
This step uses ATP.
• The amino group of 5-phosphoribosyl amine
becomes nitrogen 9 of the purine ring.
• 5, 10-Methenyl tetrahydrofolate supply the last
atom to the 5-membered ring.
• the amide of glutamine adds to carbon 4 to start
the six-membered ring portion. It becomes
nitrogen 3.
• Then condensing of carbon 8 and nitrogen 9 to
form the five-membered ring.
• the addition of carboxyl group (from carbon
dioxide) to form carbon 6 of the ring.
• The amine group of aspartate adds to the
carboxyl group with a subsequent removal of
fumarate. The amino group is now nitrogen 1 of
the final ring.
• The final atom of the purine ring, carbon 2, is
supplied by 10-Formyl tetrahydrofolate. Ring
closure produces the purine nucleotide, IMP
(inosine monophosphate). IMP can then become
either AMP or GMP via appropriate rxn
• Total 4 ATP are required for the whole process
NAD + Gln
ATP
GMP
Schematic presentation of purine synthesis
RDS- Adenylsuccinate lyse
Goes to TCA
in muscle
Regulation of Purine Nucleotide Synthesis
Regulation of Purine Biosynthesis
1- Concentration of PRPP which depends on:
• Availability of ribose 5- phosphate.
• Activity of PRPP synthase.
2-Accumulation of purine nucleotides:
• The first limiting step, PRPP amidotransferase is
synergistically inhibited by IMP & GMP binding to one
allosteric site, and AMP binding to another.
• Adenylosuccinate synthetase & IMP dehydrogenase, the
two enzymes at IMP branch point are also allosterically
regulated.
• Conversion of MPNucleotide  TPNucleotide
- For ATP, 2 systems
i- by a 2-step reactions
a) ATP-dependent transphosphorylation of AMP into ADP
ADP + ADP
AMP
*** Formation of ATP
- by phosphorylation of ADP or AMP
ATP
b) oxidative phosphorylation of ADP into ATP
ADP + Pi + O2
NADH
ATP + H2O
NAD+
this system is a compartmentalization type, i.e the enzymes catalysing the 2 rxn are linked
into a complex .The ATP which is the phosphate donor for the transphosphorylation
reaction is not the free ATP pre-existing in mitochondria, but the ATP produced by the
complex itself; on the other hand, the ADP formed as a transient intermediate in the AMPATP conversion is immediately phosphorylated to give ATP without mixing with the free
internal ADP.
ii- Conversion of mononucleotides to nucleotide di &
triphosphates
Subsequent phosphorylation of AMP & GMP by ATP, leads
to formation of di- & triphosphates catalyzed by kinases.
continuo
3-Energy sources: as seen in the reactions above, ATP is
required to synthesize GMP from XMP, while GTP is required to
synthesize AMP.
Adenylosuccinate
synthetase
PRPP
amidotransferase
IMP dehydrogenase
Synthesis of Pyrimidine Nucleotides
• pyrimidine molecules are simpler than
purines.
• their synthesis is simpler
• occurs in spleen, thymus, GI tract and testes
• Glutamine's amide nitrogen and carbon dioxide
provide atoms 2 and 3 of the pyrimidine ring via
formation of cabamoyl-PO4.
• The other four atoms of the ring are from aspartate, incorporated
followed by dehydration forming orotate derivative
• the sugar phosphate portion of the molecule is supplied by PRPP
via formation of OMP (orotate monophosphate), and subsequent
reaction leads to formation of UMP.
• UMP then acts as substrate for synthesis of other nucleotides
** Synthesis of nucleotide of pyrimidine differs
from that of purine in that:
In purine synthesis, a nucleotide is formed first
while pyrimidines are first synthesized as the free
base.
The control of pyrimidine nucleotide synthesis in
man is exerted primarily at the level of
cytoplasmic CPS II. UTP inhibits the enzyme,
competitively with ATP. PRPP activates it .
Synthesis of deoxyribonucleotides
•
•
•
•
DNA requires deoxyribonucleotides.
Conversion of purine & pyrimidine
ribonucleotides to deoxyribonucleotides occurs
only at nucleoside diphosphate level.
Nucleoside Diphosphates [NDPs] are reduced
by ribonucleotide reductase complex forming
deoxyribonucleoside diphosphates [dNDPs].
This reduction requires thioredoxin (a protein
cofactor), thioredoxin reductase (flavoproteins),
& NADPH.H+
This enzyme complex is active only when cells
are synthesizing DNA preparatory to cell
division.
GOOD LUCK
Deoxyribonucleic Acid (DNA)
• It has high molecular weight i.e molecule has high number of
nucleotides.
• In euaryotic cells, it is found chiefly in the nuclei. It wrapped around
small proteins known as histones forming bead-like structure then
organized and folded into chromatin aggregates that make up the
chromosomes
• and in procaryotic cells e.g bacteria, in the nucleoid regions.
• It contains two polynucleotide strands wound around each other
through base-pairing i.e double helix
• The backbone of each strand consists of alternating deoxyribose and
phosephate groups.
• The phosphate group bonded to the 5' carbon atom of one
deoxyribose is covalently bonded to the 3' carbon of the next.
• The two strands are "antiparallel"; that is, one strand runs 5′ to 3′
while the other runs 3′ to 5′.
Base-pair
adenine-thymine pair
guanine-cytosine pair
• The DNA strands are assembled in the 5′ to 3′ direction and, by
convention, we "read" them the same way.
• The purine or pyrimidine attached to each deoxyribose projects in
toward the axis of the helix.
• Has a cistron region i.e a sequence that contains information for a
polypeptide and several signals that are required for ribosome
function.
• Each base forms hydrogen bonds with the one directly opposite it,
forming base pairs (also called nucleotide pairs).
• adenine-thymine base pair has 2 hydrogen bonds, and guaninecytosine base pair has 3 hydrogen bonds
• guanine-cytosine base pair is stronger than the adenine-thymine base
pair
RIBONUCLEIC ACID (RNA)
• Mostly is a single-stranded molecule which can coil back on itself and
form unique and quite complex 3-D structure e.g hairpin, clover-shape.
• RNA is involved in the synthesis of proteins. "Information" is typically
passed from DNA to RNA to the resulting proteins.
• There are 3 major species :
ribosomal RNA (rRNA): 80-90%; transfer RNA
(tRNA): 15 %
messenger RNA (mRNA): 5%
• The size of the rRNA varies, but is generally less than a thousandth
the size of DNA. It is a component of ribosomes
( rRNA + proteins). The secondary structure is extraordinary complex.
Its size is designated by S (= sedimentation) value e.g 5 S, 5.8 S, 28
S.
tRNA is a small molecule consists of 65-110 nucleotides, function to carry
activated a.a to the protein synthesis site, the ribosomes. It is a stable
molecule but short-lived. There are at least 56 types in any cell. Each
recognizes a different codon for an a.a. The different tRNA that accepts
an a.a is called isoacceptor. Each carries only 1 a.a
It has a ''cloverleaf '' structure i.e consisting of a stem and 3
loops.Intrachain base pairing at some point results in double helix portion
.
One of the loop is anticodon loop. This loop has ''Anticodon zone'' which is
a triplet that base pair to mRNA during protein synthesis, and plays a role
in specifying which a.a becomes attach to the tRNA .The stem ends in
the sequence...CCA (3’end) which is the attachment site for the
a.a. It contains other determinants of which a.a is to attach to the tRNA.
An extra arm (variable loop) may also exist on the structure. tRNA made
up 15% of cellular RNA. Contain modified bases e.g 4-thioUridine,
dihydrouridine,
• mRNA is the carrier of genetic information on
the primary structure of protein from DNA, has
features allowing it to attach to ribosome and
function in protein synthesis. It is of variable size depending on the
protein size of which it codes and the cell type e.g E.coli mRNA
compose of 500-6000 nucleotides.
It is relatively short-lived, varies with protein species.
• 1 mRNA of eukaryotic codes one protein i.e it is monocistronic
whereas that of prokaryotes are polycistronic i.e contain coding
information for many polypeptide chains.