6. Nucleosides
Purine or pyrimidine base + Sugar through an N-
glycosidic linkage
Purines bind to the C1’ carbon of the sugar at their
N9 atoms
Pyrimidines bind to the C1’ carbon of the sugar at
their N1 atoms.
10. 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
11. Nucleotides
Monomers for nucleic acid polymers
Nucleoside Triphosphates are important energy carriers
(ATP, GTP)
cAMP
Important components of coenzymes
FAD, NAD+
and Coenzyme A
12. 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
15. 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
16.
17. Most of the tissues
Liver
Cytosol
Multi enzyme complex
18. Purine Nucleotide Synthesis
OH
H
H
CH2
OH OH
H H
O
α
O
2-
O3P
α-D-Ribose-5-Phosphate (R5P)
O
H
H
CH2
OH OH
H H
O α
O
2-
O3P
5-Phosphoribosyl-α-pyrophosphate (PRPP)
P
O
O
O P
O
O
O
PRPP Synthase
19. O
H
H
CH2
OH OH
H H
O α
O
2-
O3P
5-Phosphoribosyl-α-pyrophosphate (PRPP)
P
O
O
O P
O
O
O
H
NH2
H
CH2
OH OH
H H
O
β
O
2-
O3P
β-5-Phosphoribosylamine (PRA)
PRPP Glutamyl amido transferase
21. H
NH
H
CH2
OH OH
H H
O
O
2-
O3P
C
O
H2C NH2
Glycinamide Ribotide (GAR)
H2C
C
NH
O
CH
H
N
O
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
GAR formyl transferase
N
5
N
10
Methenyl (Formyl) THF
29. 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)
30. Coupling of Reactions
Hydrolyzing a phosphate from ATP is relatively easy
∆G°’= -30.5 kJ/mol
If endergonic reaction released energy into cell as heat
energy, wouldn’t be useful
Must be coupled to an exergonic reaction
When ATP is a reactant:
Part of the ATP can be transferred to an acceptor: Pi,
PPi, adenyl or adenosinyl group
ATP hydrolysis can drive an otherwise unfavorable
reaction
(synthetase; “energase”)
33. Regulation 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 Pyrophospho synthetase
PRPP Glutamyl amido transferase is activated by
PRPP levels
34. Regulation of Purine Nucleotide
Biosynthesis
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
35. Regulation 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
Total amounts of purine nucleotides controlled
Relative amounts of ATP, GTP controlled
39. 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)
40. 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.
42. 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 re-synthesized de novo !
44. Guanosine 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
Guanosine
Guanine
45.
46. 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
47. A CASE STUDY : GOUT
A 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A
PAINFUL AND SWOLLEN RIGHT GREAT TOE. ON THE
PREVIOUS NIGHT HE HAD EATEN A MEAL OF FRIED
LIVER AND ONIONS, AFTER WHICH HE MET WITH HIS
POKER GROUP AND DRANK A NUMBER OF BEERS.
HE SAW HIS DOCTOR THAT MORNING, “GOUTY
ARTHRITIS” WAS DIAGNOSED, AND SOME TESTS WERE
ORDERED. HIS SERUM URIC ACID LEVEL WAS ELEVATED
AT 8.0 mg/dL (NL < 7.0 mg/dL).
THE MAN RECALLED THAT HIS FATHER AND HIS
GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS,
OFTEN COMPLAINED OF JOINT PAIN AND SWELLING IN
THEIR FEET.
48. A CASE STUDY : GOUT
THE DOCTOR RECOMMENDED THAT THE MAN USE
NSAIDS FOR PAIN AND SWELLING, INCREASE HIS FLUID
INTAKE (BUT NOT WITH ALCOHOL) AND REST AND
ELEVATE HIS FOOT. HE ALSO PRESCRIBED
ALLOPURINOL.
A FEW DAYS LATER THE CONDITION HAD RESOLVED
AND ALLOPURINOL HAD BEEN STOPPED. A REPEAT
URIC ACID LEVEL WAS OBTAINED (7.1 mg/dL). THE
DOCTOR GAVE THE MAN SOME ADVICE REGARDING
LIFE STYLE CHANGES.
49. Gout
Impaired excretion or overproduction of uric acid
Uric acid crystals precipitate into joints (Gouty
Arthritis), kidneys, ureters (stones)
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
50.
51.
52.
53. ALLOPURINOL is a XANTHINE OXIDASE inhibitor
A substrate ANALOG is converted to an inhibitor.
In this case a “SUICIDE-INHIBITOR”
57. Lesch-Nyhan Syndrome
A defect in production or activity of HGPRT.
It is an X-linked disorder.
Causes increased level of Hypoxanthine and
Guanine (↑ in degradation to uric acid)
Also PRPP accumulates
stimulates production of Purine nucleotides (and
thereby increases their degradation)
Causes gout-like symptoms, but also neurological
symptoms spasticity, aggressiveness, self-
mutilation
First neuropsychiatric abnormality that was
attributed to a single enzyme
58.
59. 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
60. 2 ATP + HCO3
-
+ Glutamine + H2O
C
O
O PO3
-2
NH2
Carbamoyl Phosphate
2 ADP +
Glutamate +
Pi
Carbamoyl
Phosphate
Synthetase II
Pyrimidine Synthesis
66. 2 ATP + HCO3
-
+ Glutamine + H2O
C
O
O PO3
-2
NH2
Carbamoyl Phosphate
NH2
C
N
H
CH
CH2
C
COO
O
HO
O
Carbamoyl Aspartate
HN
C
N
H
CH
CH2
C
COO
O
O
Dihydroorotate
HN
C
N
H
C
CH
C
COO
O
O
Orotate
HN
C
N
C
CH
C
COO
O
O
H
H
CH2
OH OH
H H
O
O
2-
O3P
β
Orotidine-5'-monophosphate
(OMP)
HN
C
N
CH
CH
C
O
O
H
H
CH2
OH OH
H H
O
O
2-
O3P
β
Uridine Monophosphate
(UMP)
2 ADP +
Glutamate +
Pi
Carbamoyl
Phosphate
Synthetase II
Aspartate
Transcarbamoylase
(ATCase)
Aspartate
Pi
H2O
Dihydroorotase
Quinone
Reduced
Quinone
Dihydroorotate
Dehydrogenase
PRPP PPi
Orotate Phosphoribosyl
Transferase
CO2
OMP
Decarboxylase
Pyrimidine Synthesis
67. 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
68. OMP DECARBOXYLASE : THE MOST
CATALYTICALLY PROFICIENT ENZYME
FINAL REACTION OF PYRIMIDINE PATHWAY
ANOTHER MECHANISM FOR
DECARBOXYLATION
A HIGH ENERGY CARBANION INTERMEDIATE
NOT NEEDED
NO COFACTORS NEEDED !
SOME OF THE BINDING ENERGY BETWEEN OMP
AND THE ACTIVE SITE IS USED TO STABILIZE THE
TRANSITION STATE
“PREFERENTIAL TRANSITION STATE BINDING”
69.
70. 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 C4in animals
71.
72. Regulation 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
73. CPS, ATC & DHOase multi enzyme complex
OPRTase & OMP decarboxylase single functional complex.
74. Salvage : PRPP and phospho ribosyl transferase
Nucleoside phosphorylase.
75. 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
76.
77. 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
78. 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 in vitro
79.
80.
81. Thioredoxin
Physiologic reducing agent of RNR
Cys pair can swap H atoms with disulfide formed regenerate
original enzyme
Thioredoxin gets oxidized to disulfide
Oxidized Thioredoxin gets reduced by NADPH ( final electron acceptor)
mediated by thioredoxin reductase
82. 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
85. Anti-Folate Drugs
Cancer cells consume dTMP quickly for DNA
replication
Interfere with thymidylate synthase rxn to decrease dTMP
production
(fluorodeoxyuridylate – irreversible inhibitor) – also affects rapidly
growing normal cells (hair follicles, bone marrow, immune system,
intestinal mucosa)
Dihydrofolate reductase step can be stopped
competitively (DHF analogs)
Anti-Folates: Aminopterin, methotrexate, trimethoprim
86. ADENOSINE DEAMINASE DEFICIENCY
IN PURINE DEGRADATION, ADENOSINE INOSINE
ENZYME IS ADA
ADA DEFICIENCY RESULTS IN SCID
“SEVERE COMBINED IMMUNODEFICIENCY”
SELECTIVELY KILLS LYMPHOCYTES
BOTH B- AND T-CELLS
MEDIATE MUCH OF IMMUNE RESPONSE
ALL KNOWN ADA MUTANTS STRUCTURALLY PERTURB
ACTIVE SITE
87. 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
88. 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
