Transcript purine

Nucleic Acids Metabolism
Nitrogenous Bases
• Planar, aromatic, and heterocyclic
• Derived from purine or pyrimidine
• Numbering of bases is “unprimed”
Nucleic Acid Bases
Purines
Pyrimidines
Sugars
• Pentoses (5-C sugars)
• Numbering of sugars is “primed”
Sugars
D-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group
Nucleosides
• Result from linking one of the sugars with a
purine or pyrimidine base through an Nglycosidic linkage
– Purines bond to the C1’ carbon of the sugar at
their N9 atoms
– Pyrimidines bond to the C1’ carbon of the sugar at
their N1 atoms
Nucleosides
Phosphate Groups
• Mono-, di- or triphosphates
• Phosphates can be bonded to either C3 or C5
atoms of the sugar
Nucleotides
• Result from linking one or more phosphates with a
nucleoside onto the 5’ end of the molecule
through esterification
Nucleotides
• RNA (ribonucleic acid) is a polymer of
ribonucleotides
• DNA (deoxyribonucleic acid) is a polymer of
deoxyribonucleotides
• Both deoxy- and ribonucleotides contain
Adenine, Guanine and Cytosine
– Ribonucleotides contain Uracil
– Deoxyribonucleotides contain Thymine
Nucleotides
• Monomers for nucleic acid polymers
• Nucleoside Triphosphates are important
energy carriers (ATP, GTP)
• Important components of coenzymes
– FAD, NAD+ and Coenzyme A
Naming Conventions
• Nucleosides:
– Purine nucleosides end in “-sine”
• Adenosine, Guanosine
– Pyrimidine nucleosides end in “-dine”
• Thymidine, Cytidine, Uridine
• Nucleotides:
– Start with the nucleoside name from above and
add “mono-”, “di-”, or “triphosphate”
• Adenosine Monophosphate, Cytidine Triphosphate,
Deoxythymidine Diphosphate
Nucleotide Metabolism
• PURINE RIBONUCLEOTIDES: formed de novo
– i.e., purines are not initially synthesized as free bases
– First purine derivative formed is Inosine Mono-phosphate
(IMP)
• The purine base is hypoxanthine
• AMP and GMP are formed from IMP
Purine Nucleotides
• Get broken down into Uric Acid (a purine)
Buchanan (mid 1900s) showed where purine ring
components came from:
N1: Aspartate Amine
C2, C8: Formate
N3, N9: Glutamine
C4, C5, N7: Glycine
C6: Bicarbonate Ion
Purine Nucleotide Synthesis
O
COO
OOC
2-
O3P O CH2
H
O
H

H
H
C
OH
OH
OH
O3P O CH2
CH
5
ADP
+ Pi
HC
N
H
SAICAR Synthetase
CH2
C
COO
AIR
Car boxylase
ADP + Pi
H

H
H
OH
OH
H
O
Ribose-5-Phosphate
Fumarate
O
P
O
O
P
O
C
O
C
H2N
CH
5
N
C
5-Aminoimidazole Ribotide (AIR)
ADP + Pi
Transferase
O
CH
C

H
H
OH
OH
HN
O
C
ADP
+ Pi
O
O3P O CH2
H
NH2
Ribose-5-Phosphate
5-Formaminoimidazole-4-carboxamide
ribotide (FAICAR)
ATP +
Glutamine +
H2O
H2C
NH
O
C
N10-Formyl-THF
THF
O
IMP
Cyclohydrolase
O
C
OH
Glycinamide Ribotide (GAR)
GAR Transformylase
N
CH
HN
C
O
HC
C5
NH
H
H
N
NH
H2O
H
N
CH
5
C
H
OH
ADP +
Glutamate + Pi
FGAM
Synthetase
GAR Synthetase
H2C
C
H
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
N
C4
NH
O
Glycine
+ ATP
THF
C
H2N
H
-5-Phosphoribosylamine (PRA)
AICAR
Transformylase
O
NH2
H
2-
N10-FormylTHF
H
N
H 2C
O3P O CH2
5-Aminoimidazole-4-carboxamide
ribotide (AICAR)
ATP
Glutamate
+ PPi
2-
N
Ribose-5-Phosphate
AIR
Synthetase
Glutamine
+ H2O
Amidophosphoribosyl
CH
5
H2N
Ribose-5-Phosphate
5-Phosphoribosyl--pyrophosphate (PRPP)
N
C4
H2N
O
Adenylosuccinate
Lyase
O
N
HC 4
N
5-Aminoimidazole-4-(N-succinylocarboxamide)
ribotide (SAICAR)
ATP
+HCO3
O
CH
5
H2N
Ribose-5-Phosphate
Ribose
Phosphate
Pyrophosphokinase
N
C4
N
Carboxyamidoimidazole Ribotide (CAIR)
AMP
2-
Aspartate
+ ATP
H2N
-D-Ribose-5-Phosphate (R5P)
ATP
C
N
C4
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
4
CH
N
N
2-
O3P O CH2
H
H
OH
O
H
H
OH
Inosine Monophosphate (IMP)
Purine Nucleotide Synthesis
at a Glance
• ATP is involved in 6 steps
• PRPP in the first step of Purine synthesis is also a precursor for
Pyrimidine Synthesis, His and Trp synthesis
– Role of ATP in first step is unique– group transfer rather than coupling
• In second step, C1 notation changes from  to  (anomers specifying
OH positioning on C1 with respect to C4 group)
• In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)
Coupling of Reactions
• Hydrolyzing a phosphate from ATP is relatively easy
G°’= -30.5 kJ/mol
– If exergonic reaction released energy into cell as heat energy, wouldn’t be
useful
– Must be coupled to an endergonic reaction
• When ATP is a reactant:
– Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl,
adenosinyl group
– ATP hydrolysis can drive an otherwise unfavorable reaction
(synthetase)
or
Purine Biosynthetic Pathway
• Channeling of some reactions on pathway organizes and controls
processing of substrates to products in each step
– Increases overall rate of pathway and protects intermediates from
degradation
• In animals, IMP synthesis pathway shows channeling at:
– Reactions 3, 4, 6
– Reactions 7, 8
– Reactions 10, 11
IMP Conversion to AMP
IMP Conversion to GMP
Regulatory Control of Purine Nucleotide
Biosynthesis
• GTP is involved in AMP synthesis and ATP is involved in GMP
synthesis (reciprocal control of production)
• PRPP is a biosynthetically “central” molecule (why?)
– ADP/GDP levels – negative feedback on Ribose Phosphate
Pyrophosphokinase
– Amidophosphoribosyl transferase is activated by PRPP levels
– APRT activity has negative feedback at two sites
• ATP, ADP, AMP bound at one site
• GTP,GDP AND GMP bound at the other site
• Rate of AMP production increases with increasing
concentrations of GTP; rate of GMP production increases with
increasing concentrations of ATP
Regulatory Control of Purine Biosynthesis
• Above the level of IMP production:
– Independent control
– Synergistic control
– Feedforward activation by PRPP
• Below level of IMP production
– Reciprocal control
• Total amounts of purine nucleotides controlled
• Relative amounts of ATP, GTP controlled
Purine Catabolism and Salvage
• All purine degradation leads to uric acid
• Ingested nucleic acids are degraded to nucleotides by
pancreatic nucleases, and intestinal phosphodiesterases in
the intestine
• Group-specific nucleotidases and non-specific phosphatases
degrade nucleotides into nucleosides
– Direct absorption of nucleosides
– Further degradation
Nucleoside + H2O  base + ribose (nucleosidase)
Nucleoside + Pi  base + r-1-phosphate (n. phosphorylase)
NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETED.
Intracellular Purine Catabolism
• Nucleotides broken into nucleosides by action of 5’nucleotidase (hydrolysis reactions)
• Purine nucleoside phosphorylase (PNP)
–
–
–
–
Inosine  Hypoxanthine
Xanthosine  Xanthine
Guanosine  Guanine
Ribose-1-phosphate splits off
• Can be isomerized to ribose-5-phosphate
• Adenosine is deaminated to Inosine (ADA)
Intracellular Purine Catabolism
• Xanthine is the point of convergence for the
metabolism of the purine bases
• Xanthine  Uric acid
– Xanthine oxidase catalyzes two reactions
• Purine ribonucleotide degradation pathway is
same for purine deoxyribonucleotides
Adenosine Degradation
Xanthosine Degradation
• Ribose sugar gets recycled (Ribose-1-Phosphate  R-5-P )
– can be incorporated into PRPP (efficiency)
• Hypoxanthine is converted to Xanthine by Xanthine Oxidase
• Guanine is converted to Xanthine by Guanine Deaminase
• Xanthine gets converted to Uric Acid by Xanthine Oxidase
Xanthine Oxidase
• A homodimeric protein
• Contains electron transfer proteins
– FAD
– Mo-pterin complex in +4 or +6 state
– Two 2Fe-2S clusters
• Transfers electrons to O2  H2O2
– H2O2 is toxic
– Disproportionated to H2O and O2 by catalase
THE PURINE NUCLEOTIDE CYCLE
AMP + H2O  IMP + NH4+
(AMP Deaminase)
IMP + Aspartate + GTP  AMP + Fumarate + GDP + Pi
(Adenylosuccinate Synthetase)
COMBINE THE TWO REACTIONS:
Aspartate + H2O + GTP  Fumarate + GDP + Pi + NH4+
The overall result of combining reactions is deamination of Aspartate to
Fumarate at the expense of a GTP
Uric Acid Excretion
• Humans – excreted into urine as insoluble
crystals
• Birds, terrestrial reptiles, some insects –
excrete insoluble crystals in paste form
– Excess amino N converted to uric acid
• (conserves water)
• Others – further modification :
Uric Acid  Allantoin  Allantoic Acid  Urea  Ammonia
Purine Salvage
• Adenine phosphoribosyl transferase (APRT)
Adenine + PRPP  AMP + PPi
• Hypoxanthine-Guanine phosphoribosyl transferase
(HGPRT)
Hypoxanthine + PRPP  IMP + PPi
Guanine + PRPP  GMP + PPi
(NOTE: THESE ARE ALL REVERSIBLE REACTIONS)
AMP,IMP,GMP do not need to be resynthesized de
novo !
Pyrimidine Ribonucleotide Synthesis
• Uridine Monophosphate (UMP) is synthesized
first
– CTP is synthesized from UMP
• Pyrimidine ring synthesis completed first; then
attached to ribose-5-phosphate
N1, C4, C5, C6 : Aspartate
C2 : HCO3N3 : Glutamine amide Nitrogen
Pyrimidine Synthesis
O
2 ATP + HCO3- + Glutamine + H2O
C
2 ADP +
Glutamate +
Pi
O
Carbamoyl
Phosphate
Synthetase II
C
C
NH2
CH
C
N
H
PO3-2
O
PRPP
C
O
C
C
N
O
HN
O
CH
HN
PPi
2-
COO
O3P
O
Orotate Phosphoribosyl
Transferase
CH2
O
H
H
OH
OH
H

H
COO
Orotidine-5'-monophosphate
(OMP)
Orotate
Carbamoyl Phosphate
Aspartate
Reduced
Quinone
Aspartate
Transcarbamoylase
(ATCase)
O
C
CH2
CH
N
H
O
2-
CH
N
H
COO
Dihydroorotate
COO
O3P
O
CH2
CH
N
O
H2O
Dihydroorotase
Carbamoyl Aspartate
C
CH2
HN
C
O
CH
HN
C
C
C
O
O
NH2
CO2
Quinone
Pi
HO
OMP
Decarboxylase
Dihydroorotate
Dehydrogenase
O
H
H
OH
OH
H

H
Uridine Monophosphate
(UMP)
UMP Synthesis Overview
• 2 ATPs needed: both used in first step
– One transfers phosphate, the other is hydrolyzed to ADP and Pi
• 2 condensation rxns: form carbamoyl aspartate and
dihydroorotate (intramolecular)
• Dihydroorotate dehydrogenase is an intra-mitochondrial
enzyme; oxidizing power comes from quinone reduction
• Attachment of base to ribose ring is catalyzed by OPRT; PRPP
provides ribose-5-P
– PPi splits off PRPP – irreversible
UMP  UTP and CTP
• Nucleoside monophosphate kinase catalyzes transfer
of Pi to UMP to form UDP; nucleoside diphosphate
kinase catalyzes transfer of Pi from ATP to UDP to
form UTP
• CTP formed from UTP via CTP Synthetase driven by
ATP hydrolysis
– Glutamine provides amide nitrogen for C4 in
animals
Regulatory Control of Pyrimidine Synthesis
• Differs between bacteria and animals
– Bacteria – regulation at ATCase rxn
• Animals – regulation at carbamoyl phosphate synthetase
II
– UDP and UTP inhibit enzyme; ATP and PRPP activate it
– UMP and CMP competitively inhibit OMP Decarboxylase
*Purine synthesis inhibited by ADP and GDP at ribose
phosphate pyrophosphokinase step, controlling level of
PRPP  also regulates pyrimidines
Degradation of Pyrimidines
• CMP and UMP degraded to bases similarly to
purines
– Dephosphorylation
– Deamination
– Glycosidic bond cleavage
• Uracil reduced in liver, forming -alanine
– Converted to malonyl-CoA  fatty acid synthesis
for energy metabolism
Deoxyribonucleotide Formation
• Purine/Pyrimidine degradation are the same for
ribonucleotides and deoxyribonucleotides
• Biosynthetic pathways are only for ribonucleotide
production
• Deoxyribonucleotides are synthesized from
corresponding ribonucleotides
DNA vs. RNA: REVIEW
• DNA composed of deoxyribonucleotides
• Ribose sugar in DNA lacks hydroxyl group at 2’
Carbon
• Uracil doesn’t (normally) appear in DNA
– Thymine (5-methyluracil) appears instead
Formation of Deoxyribonucleotides
• Reduction of 2’ carbon done via a free radical
mechanism catalyzed by “Ribonucleotide
Reductases”
– E. coli RNR reduces ribonucleoside diphosphates (NDPs) to
deoxyribonucleoside diphosphates (dNDPs)
• Two subunits: R1 and R2
– A Heterotetramer: (R1)2 and (R2)2
Thymine Formation
• Formed by methylating deoxyuridine
monophosphate (dUMP)
• UTP is needed for RNA production, but dUTP not
needed for DNA
– If dUTP produced excessively, would cause substitution
errors (dUTP for dTTP)
• dUTP hydrolyzed by dUTPase
(dUTP diphosphohydrolase) to dUMP  methylated
at C5 to form dTMP rephosphorylate to form dTTP