bpt 114. purine and pyrimidine metabolism
DESCRIPTION
MetabolismTRANSCRIPT
Purine and Pyrimidine metabolism
BPT 114: DIGESTIVE, REPRODUCTIVE,ENDOCRINE AND URINARY SYSTEM
LECTURER;Dr. Geoffrey K. MaiyohDepartment of Medical Biochemistry
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• Purines and pyrimidines are Nitrogen containing heterocyclic compounds whose rings contain Carbon and Nitrogen.
• The planar character of purines and pyrimidines facilitates their close association or stacking which stabilizes double stranded DNA.
• They are also refereed to as Nitrogenous Bases and are a major components of nucleotides that build DNA and RNA
PURINES AND PYRIMIDINES
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Biological functions of nucleotides Building blocks of nucleic acids (DNA and RNA). Involved in energy storage, muscle contraction,
active transport, maintenance of ion gradients. Activated intermediates in biosynthesis
(e.g. UDP-glucose, S-adenosylmethionine). Components of coenzymes (NAD+, NADP+, FAD,
FMN, and CoA) Metabolic regulators:
a. Second messengers (cAMP, cGMP)b. Phosphate donors in signal transduction (ATP)
c. Regulation of some enzymes via adenylation and uridylylation
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Nitrogenous Bases• Planar, aromatic, and heterocyclic compounds• Derived from purine or pyrimidine• Numbering of bases is “unprimed”
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Nucleic Acid Bases
Purines Pyrimidines
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Sugars• Pentoses (5-C sugars)• Numbering of sugars is “primed”
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Sugars D-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group
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Nucleosides
• Result from linking one of the sugars with a purine or pyrimidine base through an N-glycosidic 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
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Nucleosides
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Phosphate Groups
• Mono-, di- or triphosphates
• Phosphates can be bonded to either C3 or C5 atoms of the sugar
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Nucleotides• Result from linking one or more phosphates with a
nucleoside onto the 5’ end of the molecule through esterification
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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 ThymineGKM/MUSOM/BPT
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Nucleotides
• Monomers for nucleic acid polymers
• Nucleoside Triphosphates are important energy carriers (ATP, GTP)
• Important components of coenzymes– FAD, NAD+ and Coenzyme A
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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 DiphosphateGKM/MUSOM/BPT
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Nucleotides
-OO
H(OH)
HH
HHO
OP
O
O-
Purine orPyrimidineBase
Phosphate
Pentose sugar
Nucleoside
Nucleotide
1'
2'3'
4'
5'β-glycosidic bond
RNA- ribose (R)DNA – deoxyribose (dR)
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NN
NNH
NH2
NNH
NH
NH
O
O
N
NH
NH2
O
H3C
NH
NH
O
O
XanthineAdenine (A)
Thymine (T)Cytosine (C)
NH
NH
O
O
Uracil (U)
NN
NNH
Purine
N
NH
Pyrimidine
1
2
3
4
5
6
3
2
16
4
57
8
9
NNH
NNH
O
NH2
Guanine (G)
NNH
NNH
O
Hypoxanthine
Nucleobase structures
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Hypoxanthine Inosine Inosinate (IMP)Xanthine Xanthosine Xanthylate (XMP)
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Two major routes for nucleotide biosynthesis
dNTPs
dNTPs
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Synthesis Pathways• For both purines and pyrimidines there are two means
of synthesis (often regulate one another)– de novo (from basic metabolites)
– salvage (recycle from pre-existing nucleotides)
Salvage Pathwayde novo PathwayGKM/MUSOM/BPT 114:DIG.END.REP.URISYS.2014
Purine nucleotides– 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
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Purine Nucleotides
• Get broken down into Uric Acid (a purine) Buchanan (mid 1900s) showed where purine ring components came from:
N1: Aspartate AmineC2, C8: FormateN3, N9: GlutamineC4, C5, N7: GlycineC6: Bicarbonate Ion
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Purine Nucleotide Synthesis
OH
H
H
CH2
OH OH
H HO
αO2-O3P
α-D-Ribose-5-Phosphate (R5P)
O
H
H
CH2
OH OH
H HO α
O2-O3P
5-Phosphoribosyl-α-pyrophosphate (PRPP)
P
O
O
O P
O
O
O
ATP
AMP
RibosePhosphatePyrophosphokinase
H
NH2
H
CH2
OH OH
H HO
βO2-O3P
β-5-Phosphoribosylamine (PRA)
AmidophosphoribosylTransferase
Glutamine + H2O
Glutamate + PPi
H
NH
H
CH2
OH OH
H HO
O2-O3P
CO
H2C NH2
Glycinamide Ribotide (GAR)
GAR Synthetase
Glycine + ATP
ADP+ Pi
H2C
CNH
O
CH
HN
O
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
H2C
CNH
O
CH
HN
HN
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
THFN10-Formyl-THF
GAR Transformylase
ATP +Glutamine +H2O
ADP +Glutamate + Pi
FGAM Synthetase
HC
CN
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole Ribotide (AIR)
ATP
ADP + Pi
AIR Synthetase
C
CN
CH
N
H2N
OOC
Ribose-5-Phosphate
4
5
Carboxyamidoimidazole Ribotide (CAIR)
ATP+HCO3
ADP + PiAIR Car boxylase
Aspartate+ ATP
ADP+ Pi
SAICAR Synthetase
AdenylosuccinateLyase
Fumarate
C
CN
CH
N
NH
Ribose-5-Phosphate
4
5
5-Formaminoimidazole-4-carboxamideribotide (FAICAR)
CH2N
O
CH
O
C
CN
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-carboxamideribotide (AICAR)
CH2N
O
C
CN
CH
N
H2N
CNH
O
HC
COO
CH2
COO
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-(N-succinylocarboxamide)ribotide (SAICAR)
THF
AICAR Transformylase
N10-Formyl-
THF
Inosine Monophosphate (IMP)
HN
HCN
C
CC
N
CH
N
O
4
5
HH
CH2
OH OH
H HOO2-O3P
IMPCyclohydrolase
H2O
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 α to β (anomers specifying OH positioning on C1 with respect to C4 group)
• In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)
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de novo Synthesis and regulation• Committed step: This is the point of no return
– Occurs early in the biosynthetic pathway– Often regulated by final product (feedback
inhibition)
X
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• 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: P i, PPi, adenyl, or adenosinyl group
– ATP hydrolysis can drive an otherwise unfavorable reaction
(synthetase; “energase”)
Coupling of Reactions
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Substrate Channeling• Substrate channeling is the process of direct
transfer of an intermediate between the active sites of two enzymes that catalyze sequential reactions in a biosynthetic pathway.
• The active sites can be located either on separate domains in a multifunctional enzyme or on separate subunits in a multienzyme complex.
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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, 11GKM/MUSOM/BPT
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IMP Conversion to AMP and GMP
Regulatory Control of Purine Nucleotide Biosynthesis
• GTP is involved in AMP synthesis and ATP is involved in GMP synthesis (reciprocal control of production)
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"We humbly place our suffering at your feet in the hope that something may change, not just for us, but for the good of the entire Church. We love these men, they love us, and in most cases, despite all efforts to renounce it, one cannot manage to give up such a solid and beautiful bond.“..Christian today, 10th June, 2014
Regulatory Control of Purine Biosynthesis
• Above the level of IMP production:– Independent control– Synergistic control
– Feed forward activation by PRPP
• Below level of IMP production– Reciprocal control
Overall• Total amounts of purine nucleotides is controlled• Relative amounts of ATP, GTP controlled
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Purine Catabolism and Salvage• All purine degradation leads to uric acid (but it might not
stop there)• 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.
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GMO phobia
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We can not even absorb whole nucleotides, how then do we absorb a gene???
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)
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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
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Major pathways of purine catabolism in animals.
ADA
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
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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 !
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Gout• Impaired excretion or overproduction of uric
acid• Uric acid crystals precipitate into joints (Gouty
Arthritis), kidneys, ureters (stones)• Lead impairs uric acid excretion – lead
poisoning from pewter drinking goblets– Fall of Roman Empire?
• Xanthine oxidase inhibitors inhibit production of uric acid, and treat gout
• Allopurinol treatment – hypoxanthine analog that binds to Xanthine Oxidase to decrease uric acid production
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ALLOPURINOL IS A XANTHINE OXIDASE INHIBITOR
A SUBSTRATE ANALOG IS CONVERTED TO AN INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”
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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 : AspartateC2 : HCO3
-
N3 : Glutamine amide Nitrogen
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2 ATP + HCO3- + Glutamine + H2O
CO
O PO3-2
NH2
Carbamoyl Phosphate
NH2
CNH
CH
CH2
C
COOO
HO
O
Carbamoyl Aspartate
HN
CNH
CH
CH2
C
COOO
O
Dihydroorotate
HN
CNH
C
CHC
COOO
O
Orotate
HN
CN
C
CHC
COOO
O
HH
CH2
OH OH
H HO
O2-O3P
β
Orotidine-5'-monophosphate(OMP)
HN
CN
CH
CHC
O
O
HH
CH2
OH OH
H HO
O2-O3P
β
Uridine Monophosphate(UMP)
2 ADP +Glutamate + Pi
CarbamoylPhosphateSynthetase II
AspartateTranscarbamoylase(ATCase)
Aspartate
Pi
H2O
Dihydroorotase
Quinone
ReducedQuinone
DihydroorotateDehydrogenase
PRPP PPi
Orotate PhosphoribosylTransferase
CO2
OMP Decarboxylase
Pyrimidine Synthesis
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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
• Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on same chain
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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
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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
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Orotic Aciduria• Caused by defect in protein chain with enzyme
activities of last two steps of pyrimidine synthesis
• Increased excretion of orotic acid in urine • Symptoms: retarded growth; severe anemia• Only known inherited defect in this pathway
(all others would be lethal to fetus)• Treat with uridine/cytidine • IN-CLASS QUESTION: HOW DOES URIDINE
AND CYTIDINE ADMINISTRATION WORK TO TREAT OROTIC ACIDURIA?
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How does UMP/UTP Cure Orotic Aciduria?
UMPSynthetase
XCarbamoylPhosphate Orotate
FeedbackInhibition
• Disease (-UMP)– No UMP/excess orotate
• Disease (+UMP)– Restore depleted UMP– Downregulate pathway via feedback inhibition (Less orotate)
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Degradation of Pyrimidines
• CMP and UMP degraded to bases similarly to purines – Dephosphorylation– Deamination– Glycosidic bond cleavage
• Uracil reduced in liver, forming β-alanine – Converted to malonyl-CoA fatty acid
synthesis for energy metabolism
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Thank you for your attention
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