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Nucleic acid metabolism
PBSCTC302 – Intermediary metabolism
M.Sc. Biochemistry
Pyrimidines Purines
 Structure of purine and pyrimidine bases
Adenine
Hypoxanthine
Guanine
XanthineUracil
Cytosine Thymine
NH3
NH3
NH3 NH3
5’methyl cytosine
CH3
Uric acid
O2 O2
 Ribose sugar, Nucleoside, Nucleotide, Nucleic acid
⍺-D-ribose sugar⍺-D-deoxyribose sugar
DeoxynucleotideDeoxynucleoside
n
Nucleotide triphosphate
H2O 2 Pi
5’ end
3’ end
Chain form
 Role of nucleotides
• Cellular concentrations of ribonucleotides are far greater than deoxyribonucleotides.
Ribonucleotides not only form RNA but also 2’-deoxyribonucleotides, required for DNA synthesis
• ATP formed by oxidative or substrate level phosphorylation acts as ‘energy-currency’ of cell
• GTP is required for capping mRNA, signal transduction, THB synthesis and microtubule formation
• Nucleotides, such as cAMP and cGMP serve as second messengers in signal transduction
• AMP is a structural component of coenzymes like CoA, FAD, NAD+, and NADP+
• Nucleotides carry activated intermediates in the synthesis of carbohydrates, lipids and conjugated
proteins, like UDP-glucose, CDP-choline, SAM, 3-phosphoadenosine 5-phosphosulphate (PAPS)
• During cell cycle, the nucleotide levels are finely regulated by concentrations of key biosynthetic
enzymes and their allosteric modulation
• Nucleotides are allosteric effectors for many important steps of intermediary metabolism
 Derivatives of Adenine
FAD
cAMP
Cysteine Pantothenate Phosphoadenosine
Coenzyme A
NAD+ NADH
 Nucleotide biosynthesis
Purine nucleotides
AMP GMP
Glycine
Glutamine
Aspartate
Pyrimidine nucleotides
Pentose sugars
UMP CMP
Carbon dioxide
Glutamine
Aspartate
John Buchnan
 De-novo purine biosynthesis
N10-formyl THF
Asp
N10-formyl THF
HCO3
-
Gln
• In 1950’s, Buchnan used isotopic tracer experiments in birds to determine the origin of N’s of purine ring
Gly
Inosine
Atom position Source
C4, C5 and N7 Glycine
N3 and N9 Glutamine
C2 and C8 C1-THF
C6 HCO3
-
N1 Aspartate
Purine nucleus
 De-novo purine biosynthesis
5-phosphoribosyl
1-pyrophosphate (PRPP)
5-phospho-β-D-ribosylamine
Glu-PRPP
amidotransferase
Gln Glu
+ H2O + PPi
1
Glycinamide
ribonucleotide (GAR)
GAR synthetase
Gly ADP
+ ATP + Pi
Mg2+
2
Formyl glycinamide
ribonucleotide (FGAR)
GAR formyltransferase
N10-Formyl
THF
3
THF
Formyl glycinamidine
ribonucleotide (FGAM)
Gln, ATP
Glu, ADP + Pi
FGAM synthetase 4
5-Aminoimidazole
ribonucleotide (AIR)
ADP + Pi ATPH2O
AIR synthetase
5
AIR carboxylase
ADP + Pi ATP
HCO3
-
Mg2+
Carboxylamino
imidazole
ribonucleotide (CAIR)
6
5-Aminoimidazole-
4-(N-succinylcarboxyamide)
ribonucleotide (SAICAR)
SAICAR synthetase
ADP + Pi ATP
Asp
7
 De-novo purine biosynthesis
Fumarate
Adenylosuccinate/
SCAIR lyase
5-aminoimidazole-
4-carboxyamide
ribonucleotide (AICAR)
8
SAICAR
N10-Formyl THF THF
AICAR
transformylase
N-formylaminoimidazole-
4-carboxyamide
ribonucleotide (FAICAR)
9
H2O
IMP synthase
Inosinate (IMP)
10
Inosinate (IMP)
 Formation of AMP and GMP from IMP branch point
Adenosuccinate Adenylate (AMP)
Adenylosuccinate
lyase
Fumarate
Xanthylate Guanylate (GMP)
GMP synthetase
Mg2+
 De-novo pyrimidine biosynthesis
Gln Glu + Pi
Carbamic acid
Carbamoyl
synthetase II
Pyrimidine
Bicarbonate
ATP ADP
Carboxyphosphate
Carbamoyl
synthetase II
Asp
Pi
Carbamoylaspartate
Aspartate
transcarbomylase
Dihydroorotate
H2O H+
Dihydroortase
Orotate
NADH
+ H+ NAD+
Dihydroortate
dehydrogenase
ATP ADP
Carbamoyl phosphate
Carbamoyl
synthetase II
 De-novo pyrimidine biosynthesis
Orotate
Orotidylate
PPi
Orotate phospho
ribosyltransferase
Uridylate (UMP)
H+
CO2
Orotidylate
decarboxylase
Cytidylate (CMP)
Glu Gln
+ ADP ATP
+ Pi
Cytidylate
synthetase
5-Phosphoribosyl-1-pyrophosphateRibose-5-phosphate
PRPP synthetase
ATP AMP
 Formation of thymidylate
• The thymine nucleotides are derived from dUMP, which in E.coli is derived from dUTP. In animal
cells, dCMP deaminase is induced before DNA synthesis begins for dTMP synthesis via dUMP.
dCMP dUMP
dCMP deaminase
H2O NH3
DHF reductase
THF
Ser
dTMP
N7, N8-DHFN5, N10-
Methylene THF
Thymidylate synthase
Gly + H2O
Serine hydroxymethyl
transferase
 Regulation of de novo purine biosynthesis
5-Phosphoribosyl 1-pyrophosphate
5-Phosphoribosyl amine
FGAR
GAR
FGAM
AIR
CAI
R
SCAIR
AICAR
FAICA
R
Inosinate Adenylosuccinate ADPAMP ATPGDPGTP GMP Xanthylate
• On demand substrate
channeling: ‘Purinosome’
complexes comprising enzyme
modules are formed when de
novo purine synthesis is
required by cell
• Negative regulation:
Synergistic feedback
inhibition of commitment
step by nucleotide end-
products shuts de novo
purine synthesis
• PRPP is a positive regulator.
Its consumption shuts de novo
purine synthesis
• Rate limiting step: Like purine synthesis, the initial reaction, catalyzed by carbamoyl phosphate synthetase II,
is the rate limiting step of the pyrimidine synthesis pathway. However, in E. coli, it is the second reaction,
catalyzed by aspartate transcarbomylase, which controls the rate of pathway. This allosteric enzyme has a
catalytic and a regulatory domain. While the catalytic domain can act independent of regulatory domain, the
presence of regulatory domain senses CTP concentration and decreases the affinity of aspartate binding to
catalytic subunit
 Regulation of de novo pyrimidine biosynthesis
• Substrate channeling: Carbamoyl synthetase II enzyme has three regions- first responsible for synthesis of
carbamic acid, second for release of ammonia from glutamine and third a channel to connect the two. Also,
the first three activities of pathway are catalyzed by same 215 kDa protein molecule comprising CPS II,
aspartate transcarbomylase and dihydoorotase modules, allowing efficiency by limiting diffusion of
intermediates. Similarly, last two activities: orotidylate dehydrogenase and orotidylate pyrophosphorylase
are catalyzed by same polypeptide
 Catabolism of GMP to uric acid
Guanosine monophosphate
GMP 5’-nucleotidase
Guanosine
H2O Pi
Guanine
Purine nucleoside
phosphorylase (PNP) /
Nucleosidase
H2O + Pi R-5-P
Xanthine
Deaminase
H2O
Pi
Uric acid
H2O2 H2O + O2
Xanthine
dehydrogenase
Xanthine
oxidase
NADH + H+ H2O + NAD+
 Catabolism of AMP to uric acid
Adenosine monophosphate (AMP) Inosine monophosphate (IMP)
AMP deaminase
H2O NH4
+
Adenosine
AMP 5’-nucleotidase
Pi H2O
Inosine
IMP 5’-nucleotidase
H2O Pi
Xantine
H2O + O2 H2O2
+ +
NAD+ NADH + H+
Xanthine oxidase
Uric acid
Xanthine oxidase
H2O + O2 H2O2
+ +
NAD+ NADH + H+Hypoxantine
PNP
H2O + Pi
R-5-P
R-5-P H2O + Pi
PNP
H2O
Allantoinase
Allantoinate
(Some bony fishes)
 Catabolism of uric acid to ammonia
Uric acid
(Primates, birds, reptiles, insects)
Allantoin
(Most mammals;turtles; some insects; gastropods)
½ O2 + H2O CO2
Urate oxidase
Excreted by:
4 NH3
Ammonia
(Plants; crustaceans; many marine vertebrates)
Urease
2 CO2 2 H2O
2 H2O
2 Urea
(Amphibians, cartilaginous fishes, marine vertebrates)
Glycolate
Allantoicase
+
 Catabolism of pyrimidines
H2O
NH3
Cytosine
Uracil
Thymine
H2O
H2O
Carbamoyl-β-alanine
Carbamoyl-β-aminoisobutyrate
Dihyropyrimidinase
Dihyropyrimidinase
NH3
⍺-Ketoglu Glu
Methylmalonyl
semialdehyde
Malonate
Aminotransferase
Aminotransferase
NH4
+ + HCO3
-
NH4
+ + HCO3
-
Ureidopropionase
Ureidopropionase
β-aminoisobutyrate
β-alanine
NADPH + H+ NADP+
NADPH + H+ NADP+
Dihyrouracil
dehydrogenase
Dihyrouracil
dehydrogenase
Dihyrouracil
Dihyrothymine
Adenine GuanineHypoxanthine
 Purine
salvage
Inosinate (IMP) Guanylate (GMP)
HGPRT
PRPP
PPi
Adenosine
PRPP
PPi
APRT
Adenylate (AMP)
Inosine Guanosine
 Syndromes or diseases due to defects in degradation of purine nucleotides
1. GOUT- Gout is a common condition due to high blood and tissue concentrations of
uric acid caused by deregulation of de novo purine biosynthesis. In gout, precipitation
of sodium urate in kidneys and regions of body with temperature below 37 ℃, like
joints and extremities results in complications in renal handling and inflamed, painful
and arthritic joints. A combinatorial therapy involves taking diet low in nucleotides
(avoiding red meat, beer and dried beans) and taking drugs such as allopurinol (a
hypoxanthine analog that acts as suicide inhibitor of xanthine oxidase), anticancer and
antihyperuricemic drugs. Gout may also result from faulty carbohydrate metabolism,
wherein deficiency of glucose-6-phosphatase (von Gierke’s disease) results in
accumulation of ribose-5-phosphate (R-5-P) instead of glucose. R-5-P leads to excess
5-Phosphoribosyl-1-pyrophosphate (PRPP) which stimulates purine synthesis, thus
producing more uric acid. Gout is more common in men. In women, oestrogen
promotes uric acid excretion
Swollen and
inflammed
joints
Uric acid
crystals
© Healthwise, Incorporated
2. LESCH NYHAN SYNDROME (LNS) – An X-linked recessive genetic disease caused due to mutations in
HGPRTase gene resulting in severe deficiency or complete lack in activity of HGPRTase (hypoxanthine
guanine phosphoribosyl transferase) which salvages guanine and hypoxanthine. If de novo pathway is
dysfunctional, AMP can be converted to GMP via IMP by APRTase (adenine phosphoribosyl transferase).
In LNS, rather than being salvaged, A and G are broken down, leading to excess uric acid. Patients excrete
4-5 times as much uric acid as gout patients do. Besides, neurological problems like spasticity, mental
retardation and self mutilation ensue, due to imbalanced purine nucleotide concentrations during CNS
development
3. Immunodeficiency diseases (SCID-SEVERE COMBINED IMMUNODEFICIENCY DISEASES) – SCID is due to
defects in purine nucleoside degradation due to a range of genetic mutations in enzymes of purine
catabolism and salvage pathways. Adenosine deaminase (ADA) and Purine nucleoside phosphorylase
(PNP) deficiency causes SCID. It is also called as the bubble boy disease due to lack of immune protection
and neurological defects
 Syndromes or diseases due to defects in degradation of purine nucleotides
 Pyrimidine salvage
Cytidine
deaminase
H2O
NH3
Zn2+
Uridine
Cytidine
Gln
+ ATP
Glu
+ ADP + Pi
CTP
synthase
Cytidine diphosphate (CDP)
Cytidylate
kinase
Nucleotide
diphosphate
phosphatase
ATP ADP
Pi H2O
UDP
ATP ADP
Pi H2O
Mg2+
Ca2+
Cytidine
kinase
5’ nucleotidase
Cytidylate (CMP)
ATP ADP
ATP ADP
Pi H2O
Mg2+
Ca2+
UMP
Pi H2O
Cytidine triphosphate (CTP)
ATP ADP
Pi H2O
Nucleoside
triphosphate
phosphatase
UTP
ATP ADP
H2O Pi
Mg2+
Ca2+
Nucleoside
diphosphate
kinase
Ca2+Apyrase
PPi
2H2O
 Formation of deoxy derivatives of nucleotides
Ribonucleotide reductase
Thioredoxin
reductase
• Ribonucleotide diphosphates are converted to 2’ deoxy-ribonucleotides by ribonucleotide diphosphate
reductase (RDR), an enzyme complex, comprising two B1 and two B2 subunits. It is active only in
dividing cells. It is subject to complex allosteric control by nucleotide triphosphates. The reaction
requires a small protein thioredoxin with two free sulfhydryl groups positioned in such a way as to form
a disulphide bond. Another enzyme, thioredoxin reductase regenerates reduced thioredoxin using
FADH2 and NADPH.
Thioredoxin
SHSH
Thioredoxin
SS
NADPH + H+NADP+
Ribonucleoside diphosphate
(ADP, GDP, CDP, UDP)
2’ deoxy-ribonucleoside diphosphate
(dADP, dGDP, dCDP, dUDP)
 Formation of nucleoside di and tri-phosphates
• Nucleoside monophosphates are converted to their di- and tri-phosphate derivatives by
phosphorylation reactions catalyzed by nucleoside monophosphate kinases (NMP) and nucleoside
diphosphate kinases (NDP) using ATP.
Nucleoside monophosphate
Nucleoside diphosphate
Nucleoside diphosphate
ATP
ADP
ATP
ADP
NMP kinase
NDP kinase
 Resources
• Principles of Biochemistry by Horton, Moran, Scrimgeour, Perry and
Rawn
• Biochemistry: A case oriented approach by Montgomery, Conway,
Spector and Chappell
• Biochemistry by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer

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Nucleotide metabolism

  • 1. Nucleic acid metabolism PBSCTC302 – Intermediary metabolism M.Sc. Biochemistry
  • 2. Pyrimidines Purines  Structure of purine and pyrimidine bases Adenine Hypoxanthine Guanine XanthineUracil Cytosine Thymine NH3 NH3 NH3 NH3 5’methyl cytosine CH3 Uric acid O2 O2
  • 3.  Ribose sugar, Nucleoside, Nucleotide, Nucleic acid ⍺-D-ribose sugar⍺-D-deoxyribose sugar DeoxynucleotideDeoxynucleoside n Nucleotide triphosphate H2O 2 Pi 5’ end 3’ end Chain form
  • 4.  Role of nucleotides • Cellular concentrations of ribonucleotides are far greater than deoxyribonucleotides. Ribonucleotides not only form RNA but also 2’-deoxyribonucleotides, required for DNA synthesis • ATP formed by oxidative or substrate level phosphorylation acts as ‘energy-currency’ of cell • GTP is required for capping mRNA, signal transduction, THB synthesis and microtubule formation • Nucleotides, such as cAMP and cGMP serve as second messengers in signal transduction • AMP is a structural component of coenzymes like CoA, FAD, NAD+, and NADP+ • Nucleotides carry activated intermediates in the synthesis of carbohydrates, lipids and conjugated proteins, like UDP-glucose, CDP-choline, SAM, 3-phosphoadenosine 5-phosphosulphate (PAPS) • During cell cycle, the nucleotide levels are finely regulated by concentrations of key biosynthetic enzymes and their allosteric modulation • Nucleotides are allosteric effectors for many important steps of intermediary metabolism
  • 5.  Derivatives of Adenine FAD cAMP Cysteine Pantothenate Phosphoadenosine Coenzyme A NAD+ NADH
  • 6.  Nucleotide biosynthesis Purine nucleotides AMP GMP Glycine Glutamine Aspartate Pyrimidine nucleotides Pentose sugars UMP CMP Carbon dioxide Glutamine Aspartate
  • 7. John Buchnan  De-novo purine biosynthesis N10-formyl THF Asp N10-formyl THF HCO3 - Gln • In 1950’s, Buchnan used isotopic tracer experiments in birds to determine the origin of N’s of purine ring Gly Inosine Atom position Source C4, C5 and N7 Glycine N3 and N9 Glutamine C2 and C8 C1-THF C6 HCO3 - N1 Aspartate Purine nucleus
  • 8.  De-novo purine biosynthesis 5-phosphoribosyl 1-pyrophosphate (PRPP) 5-phospho-β-D-ribosylamine Glu-PRPP amidotransferase Gln Glu + H2O + PPi 1 Glycinamide ribonucleotide (GAR) GAR synthetase Gly ADP + ATP + Pi Mg2+ 2 Formyl glycinamide ribonucleotide (FGAR) GAR formyltransferase N10-Formyl THF 3 THF Formyl glycinamidine ribonucleotide (FGAM) Gln, ATP Glu, ADP + Pi FGAM synthetase 4 5-Aminoimidazole ribonucleotide (AIR) ADP + Pi ATPH2O AIR synthetase 5 AIR carboxylase ADP + Pi ATP HCO3 - Mg2+ Carboxylamino imidazole ribonucleotide (CAIR) 6 5-Aminoimidazole- 4-(N-succinylcarboxyamide) ribonucleotide (SAICAR) SAICAR synthetase ADP + Pi ATP Asp 7
  • 9.  De-novo purine biosynthesis Fumarate Adenylosuccinate/ SCAIR lyase 5-aminoimidazole- 4-carboxyamide ribonucleotide (AICAR) 8 SAICAR N10-Formyl THF THF AICAR transformylase N-formylaminoimidazole- 4-carboxyamide ribonucleotide (FAICAR) 9 H2O IMP synthase Inosinate (IMP) 10
  • 10. Inosinate (IMP)  Formation of AMP and GMP from IMP branch point Adenosuccinate Adenylate (AMP) Adenylosuccinate lyase Fumarate Xanthylate Guanylate (GMP) GMP synthetase Mg2+
  • 11.  De-novo pyrimidine biosynthesis Gln Glu + Pi Carbamic acid Carbamoyl synthetase II Pyrimidine Bicarbonate ATP ADP Carboxyphosphate Carbamoyl synthetase II Asp Pi Carbamoylaspartate Aspartate transcarbomylase Dihydroorotate H2O H+ Dihydroortase Orotate NADH + H+ NAD+ Dihydroortate dehydrogenase ATP ADP Carbamoyl phosphate Carbamoyl synthetase II
  • 12.  De-novo pyrimidine biosynthesis Orotate Orotidylate PPi Orotate phospho ribosyltransferase Uridylate (UMP) H+ CO2 Orotidylate decarboxylase Cytidylate (CMP) Glu Gln + ADP ATP + Pi Cytidylate synthetase 5-Phosphoribosyl-1-pyrophosphateRibose-5-phosphate PRPP synthetase ATP AMP
  • 13.  Formation of thymidylate • The thymine nucleotides are derived from dUMP, which in E.coli is derived from dUTP. In animal cells, dCMP deaminase is induced before DNA synthesis begins for dTMP synthesis via dUMP. dCMP dUMP dCMP deaminase H2O NH3 DHF reductase THF Ser dTMP N7, N8-DHFN5, N10- Methylene THF Thymidylate synthase Gly + H2O Serine hydroxymethyl transferase
  • 14.  Regulation of de novo purine biosynthesis 5-Phosphoribosyl 1-pyrophosphate 5-Phosphoribosyl amine FGAR GAR FGAM AIR CAI R SCAIR AICAR FAICA R Inosinate Adenylosuccinate ADPAMP ATPGDPGTP GMP Xanthylate • On demand substrate channeling: ‘Purinosome’ complexes comprising enzyme modules are formed when de novo purine synthesis is required by cell • Negative regulation: Synergistic feedback inhibition of commitment step by nucleotide end- products shuts de novo purine synthesis • PRPP is a positive regulator. Its consumption shuts de novo purine synthesis
  • 15. • Rate limiting step: Like purine synthesis, the initial reaction, catalyzed by carbamoyl phosphate synthetase II, is the rate limiting step of the pyrimidine synthesis pathway. However, in E. coli, it is the second reaction, catalyzed by aspartate transcarbomylase, which controls the rate of pathway. This allosteric enzyme has a catalytic and a regulatory domain. While the catalytic domain can act independent of regulatory domain, the presence of regulatory domain senses CTP concentration and decreases the affinity of aspartate binding to catalytic subunit  Regulation of de novo pyrimidine biosynthesis • Substrate channeling: Carbamoyl synthetase II enzyme has three regions- first responsible for synthesis of carbamic acid, second for release of ammonia from glutamine and third a channel to connect the two. Also, the first three activities of pathway are catalyzed by same 215 kDa protein molecule comprising CPS II, aspartate transcarbomylase and dihydoorotase modules, allowing efficiency by limiting diffusion of intermediates. Similarly, last two activities: orotidylate dehydrogenase and orotidylate pyrophosphorylase are catalyzed by same polypeptide
  • 16.  Catabolism of GMP to uric acid Guanosine monophosphate GMP 5’-nucleotidase Guanosine H2O Pi Guanine Purine nucleoside phosphorylase (PNP) / Nucleosidase H2O + Pi R-5-P Xanthine Deaminase H2O Pi Uric acid H2O2 H2O + O2 Xanthine dehydrogenase Xanthine oxidase NADH + H+ H2O + NAD+
  • 17.  Catabolism of AMP to uric acid Adenosine monophosphate (AMP) Inosine monophosphate (IMP) AMP deaminase H2O NH4 + Adenosine AMP 5’-nucleotidase Pi H2O Inosine IMP 5’-nucleotidase H2O Pi Xantine H2O + O2 H2O2 + + NAD+ NADH + H+ Xanthine oxidase Uric acid Xanthine oxidase H2O + O2 H2O2 + + NAD+ NADH + H+Hypoxantine PNP H2O + Pi R-5-P R-5-P H2O + Pi PNP
  • 18. H2O Allantoinase Allantoinate (Some bony fishes)  Catabolism of uric acid to ammonia Uric acid (Primates, birds, reptiles, insects) Allantoin (Most mammals;turtles; some insects; gastropods) ½ O2 + H2O CO2 Urate oxidase Excreted by: 4 NH3 Ammonia (Plants; crustaceans; many marine vertebrates) Urease 2 CO2 2 H2O 2 H2O 2 Urea (Amphibians, cartilaginous fishes, marine vertebrates) Glycolate Allantoicase +
  • 19.  Catabolism of pyrimidines H2O NH3 Cytosine Uracil Thymine H2O H2O Carbamoyl-β-alanine Carbamoyl-β-aminoisobutyrate Dihyropyrimidinase Dihyropyrimidinase NH3 ⍺-Ketoglu Glu Methylmalonyl semialdehyde Malonate Aminotransferase Aminotransferase NH4 + + HCO3 - NH4 + + HCO3 - Ureidopropionase Ureidopropionase β-aminoisobutyrate β-alanine NADPH + H+ NADP+ NADPH + H+ NADP+ Dihyrouracil dehydrogenase Dihyrouracil dehydrogenase Dihyrouracil Dihyrothymine
  • 20. Adenine GuanineHypoxanthine  Purine salvage Inosinate (IMP) Guanylate (GMP) HGPRT PRPP PPi Adenosine PRPP PPi APRT Adenylate (AMP) Inosine Guanosine
  • 21.  Syndromes or diseases due to defects in degradation of purine nucleotides 1. GOUT- Gout is a common condition due to high blood and tissue concentrations of uric acid caused by deregulation of de novo purine biosynthesis. In gout, precipitation of sodium urate in kidneys and regions of body with temperature below 37 ℃, like joints and extremities results in complications in renal handling and inflamed, painful and arthritic joints. A combinatorial therapy involves taking diet low in nucleotides (avoiding red meat, beer and dried beans) and taking drugs such as allopurinol (a hypoxanthine analog that acts as suicide inhibitor of xanthine oxidase), anticancer and antihyperuricemic drugs. Gout may also result from faulty carbohydrate metabolism, wherein deficiency of glucose-6-phosphatase (von Gierke’s disease) results in accumulation of ribose-5-phosphate (R-5-P) instead of glucose. R-5-P leads to excess 5-Phosphoribosyl-1-pyrophosphate (PRPP) which stimulates purine synthesis, thus producing more uric acid. Gout is more common in men. In women, oestrogen promotes uric acid excretion Swollen and inflammed joints Uric acid crystals © Healthwise, Incorporated
  • 22. 2. LESCH NYHAN SYNDROME (LNS) – An X-linked recessive genetic disease caused due to mutations in HGPRTase gene resulting in severe deficiency or complete lack in activity of HGPRTase (hypoxanthine guanine phosphoribosyl transferase) which salvages guanine and hypoxanthine. If de novo pathway is dysfunctional, AMP can be converted to GMP via IMP by APRTase (adenine phosphoribosyl transferase). In LNS, rather than being salvaged, A and G are broken down, leading to excess uric acid. Patients excrete 4-5 times as much uric acid as gout patients do. Besides, neurological problems like spasticity, mental retardation and self mutilation ensue, due to imbalanced purine nucleotide concentrations during CNS development 3. Immunodeficiency diseases (SCID-SEVERE COMBINED IMMUNODEFICIENCY DISEASES) – SCID is due to defects in purine nucleoside degradation due to a range of genetic mutations in enzymes of purine catabolism and salvage pathways. Adenosine deaminase (ADA) and Purine nucleoside phosphorylase (PNP) deficiency causes SCID. It is also called as the bubble boy disease due to lack of immune protection and neurological defects  Syndromes or diseases due to defects in degradation of purine nucleotides
  • 23.  Pyrimidine salvage Cytidine deaminase H2O NH3 Zn2+ Uridine Cytidine Gln + ATP Glu + ADP + Pi CTP synthase Cytidine diphosphate (CDP) Cytidylate kinase Nucleotide diphosphate phosphatase ATP ADP Pi H2O UDP ATP ADP Pi H2O Mg2+ Ca2+ Cytidine kinase 5’ nucleotidase Cytidylate (CMP) ATP ADP ATP ADP Pi H2O Mg2+ Ca2+ UMP Pi H2O Cytidine triphosphate (CTP) ATP ADP Pi H2O Nucleoside triphosphate phosphatase UTP ATP ADP H2O Pi Mg2+ Ca2+ Nucleoside diphosphate kinase Ca2+Apyrase PPi 2H2O
  • 24.  Formation of deoxy derivatives of nucleotides Ribonucleotide reductase Thioredoxin reductase • Ribonucleotide diphosphates are converted to 2’ deoxy-ribonucleotides by ribonucleotide diphosphate reductase (RDR), an enzyme complex, comprising two B1 and two B2 subunits. It is active only in dividing cells. It is subject to complex allosteric control by nucleotide triphosphates. The reaction requires a small protein thioredoxin with two free sulfhydryl groups positioned in such a way as to form a disulphide bond. Another enzyme, thioredoxin reductase regenerates reduced thioredoxin using FADH2 and NADPH. Thioredoxin SHSH Thioredoxin SS NADPH + H+NADP+ Ribonucleoside diphosphate (ADP, GDP, CDP, UDP) 2’ deoxy-ribonucleoside diphosphate (dADP, dGDP, dCDP, dUDP)
  • 25.  Formation of nucleoside di and tri-phosphates • Nucleoside monophosphates are converted to their di- and tri-phosphate derivatives by phosphorylation reactions catalyzed by nucleoside monophosphate kinases (NMP) and nucleoside diphosphate kinases (NDP) using ATP. Nucleoside monophosphate Nucleoside diphosphate Nucleoside diphosphate ATP ADP ATP ADP NMP kinase NDP kinase
  • 26.  Resources • Principles of Biochemistry by Horton, Moran, Scrimgeour, Perry and Rawn • Biochemistry: A case oriented approach by Montgomery, Conway, Spector and Chappell • Biochemistry by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer

Editor's Notes

  1. IMP and GMP are oxypurines while AMP is aminopurine. Instead of T, RNA contain U (demethylated T). C is complimentary to G while T is opposite A. Hydrogen bonds have 8 kcal/mole energy. DNA double helix is thus tight enough to exist at body temperature but not stable enough not to be pulled apart/denatured when certain conditions dictate.
  2. N-Glycosidic bond is formed between the base-N (N-1 of pyrimidines and N-9 of purines) and sugar-C (C1).
  3. Antimetabolite theory proposed by Elions and Hitchings (1951) suggests that rapid growth of cells can be inhibited by preventing the utilization of essential nutrients. They synthesized, tested and identified purine analogs like 6-Thioguanine, 2,6-Diaminopurine and 6-Mercaptopurine which curtailed bacterial and cancer growth. Drugs that block de novo pathways of nucleotide synthesis have been used as antitumor and antiviral agents. Cancer is characterized by greatly increased concentration of nucleotide triphosphates (3.6 nmol per 106 Ehrlich tumor cells as compared to 4 pmol per 106 normal cells). In contrast, hypoxia is associated with increased concentrations of nucleotide mono and diphosphates PAPS is used as a sulfate donor for synthesis of sulfated biomolecules like proteoglycans and sulfatides
  4. Total cellular concentration of adenine nucleotides is four to six times that of guanine nucleotides
  5. Nucleotides are synthesized from 5-C sugars and amino acids.
  6. John Buchnan characterized one of the first examples of mechanism based inactivator (suicide inactivator)- Azaserine, a glutamine analogue which inhibits glutamine amidotransferase.
  7. PRPP is a precursor common to both purine and pyrimidine biosynthesis. Purine biosynthesis is a ten step process involving formation of common intermediate Inosine All enzymes involved in Inosine synthesis are cytosolic Purine synthesis requires intensive energy investment. Six mole equivalent of ATP are utilized per mole of IMP synthesized
  8. Inosine serves as common precursor for AMP and GMP.
  9. De novo nucleotide biosynthesis requires a common precursor 5-Phosphoribosyl-1-pyrophosphate which is synthesized from a ribose-5-phosphate from pentose phosphate pathway. But, unlike synthesis of purine nucleotides, the pyrimidine ring is formed before PRPP moiety is attached.
  10. Step 1 is the formation of 5-phosphoribosylamine from PRPP and Glutamine. It is the commitment step and is allosterically regulated. The activation by enzyme Glu-PRPP amidotransferase by PRPP involves its active monomeric form of 135 kDa while its feedback inhibition by end products of the pathway-IMP, GMP and AMP involve its dimerization. Steps 2-10 are not known to be regulated. When purine synthesis is required by the cell, the enzymes of the purine synthesis pathway associate with one another to form purinosome complexes in order to promote substrate channeling. A trifunctional protein with three activities : GAR synthetase, GAR transformylase and AIR synthetase besides two bifunctional proteins, first one with AIR carboxylase and SAICAR synthetase while second one with AICAR transformylase and IMP synthase activities clubbed together operate in the inosine synthesis pathway. Purine and pyrimidine biosynthesis parallel one another mole for mole. PRPP biosynthesis is feedback inhibited by both purine and pyrimidine nucleotides. Defects in regulation of purine synthesis leads to accumulation of purine nucleotides and their catabolic end product, uric acid causing Gout
  11. CPS enzyme has two isoforms I and II. CPS I is mitochondrial, used in urea synthesis, activated by N-acetyl glutamate and uses ammonia as N-source while CPS II is cytoplasmic, used for pyrimidine synthesis, inhibited by CTP and uses glutamine as N-source Orotic aciduria, a genetically inherited disease due to lack of orotidylate pyrophosphorylase (OPP) and orotidylate decarboxylase (ODC) activities leads to accumulation of orotic acid, which is excreted in urine. Treatment involves oral administration of uridine
  12. Guanine and hypoxanthine have two fates: conversion to 5’ ribonucleotides or xanthine. Oxidation of hypoxanthine and xanthine to uric acid is catalyzed by xanthine oxidase. This enzyme is found in liver and intestinal mucosa. It is a metalloflavoprotein that contains FAD, molybdenum and nonheme iron. Electrons from the substrates are passed to molybdenum, FAD, iron and finally molecular oxygen which is reduced to hydrogen peroxide.
  13. Nucleotidases are enzymes that convert nucleotides to nucleosides. They are also refered to as phosphomonoesterases. Second reaction catalyzed by purine nucleoside phosphorylase is mechanistically similar to glycogen phosphorylase catalyzed removal of sugar 1-phosphate using inorganic phosphate. During intense exercise, AMP deaminase converts AMP to IMP, releasing ammonia. Decreasing AMP levels shifts equilibrium of adenylate kinase to produce more ATP for muscle contraction. IMP can be recycled to AMP via adenylosuccinate intermediate. The reaction uses a molecule each of water, GTP, and aspartate and releases fumarate with the generation of AMP from IMP.
  14. Purines are catabolized to uric acid which is excreted at a rate of about 0.6 g per 24 h by a healthy adult human. Humans are only mammals unable to breakdown uric acid to allantoin due to non-functional urate oxidase gene which encodes the uricase enzyme. Loss of uricase in hominids (humans, chimpanzees and gorillas) during evolution is proposed to be advantageous since uric acid is powerful antioxidant and scavanger of singlet oxygen and radicals, thus prolonging life and decreasing age related cancer rates. Urate oxidase is formulated as a protein drug named rasburicase for the treatment of acute hyperuricemia in patients receiving chemotherapy.
  15. Catabolism of pyrimidines occurs in liver using NADPH as reducing agent followed by ring opening, and hydrolysis to carbamoyl derivatives which are further deaminated, decarboxylated and transaminated to yield malonate and methylmalonate. Catabolic reactions are reverse of anabolic ones except that intermediates are not orotic acid derivatives.
  16. Many organisms salvage purine and pyrimidine bases to their mononucleotides. Recycling of products of nucleotide metabolism from diet and degradative pathways, occurs mainly in the extrahepatic tissue, in contrast to de novo pathway which occurs in liver. De novo synthesis of nucleotides does not take place in brain and erythrocytes, making salvage pathway indispensable for them The salvage pathway requires no ATP and accounts for 90% of nucleotide biosynthesis. HGPRT is the most important salvage enzyme. Its regulation occurs by competitive inhibition by IMP and GMP. APRT is inhibited by AMP.
  17. Excess pyrimidine degradation occurs during fast cell destruction as in leukemia or in patients undergoing radiation therapy. Fortunately, degradation products of pyrimidine catabolism (malonate and methylmalonate) are more soluble than those produced by purine degradation. Therefore, they are easily excreted in urine and there is no known complication analogous to gout caused due to pyrimidine catabolism. Allopurinol is oxidized by Xanthine dehydrogenase/oxidase to oxypurinol, an inhibitor of Xanthine dehydrogenase
  18. LNS patients may also have mutations in APRTase and IMP dehydrogenase gene. As GTP and Hypoxanthine are not salvaged, PRPP is not consumed, and de novo synthesis of purine nucleotides is not shut down. GTP is a precursor of Tetrahydrobiopterin, a precursor for neurotransmitter biosynthesis. Lack of GTP therefore has neurological manifestations. Treatment of LNS patient with allopurinol decreases the amount of uric acid formed.
  19. Nucleoside triphosphate phosphatase and nucleoside diphosphate kinase enzyme pair catalyzes the reversible conversion of CDP and CTP as well as UDP and UTP. Similarly, cytidylate/uridylate kinase and nucleoside diphosphate phosphatase catalyze reversible conversion of CDP and CMP as well as UDP and UMP.
  20. RDR