Transcript No Slide Title

Nucleotides: Synthesis
and Degradation
Roles of Nucleotides
•Precursors to nucleic acids (genetic material and non-protein
enzymes).
•Currency in energy metabolism (eg. ATP, GTP).
•Carriers of activated metabolites for biosynthesis
(eg. CDP, UDP).
•Structural moieties of coenzymes (eg. NAD, CoA).
•Metabolic regulators and signal molecules (eg. cAMP,
cGMP, ppGpp).
Biosynthetic routes: De novo and salvage pathways
De novo pathways
Almost all cell types have the ability to synthesize purine and
pyrimidine nucleotides from low molecular weight precursors in
amounts sufficient for their own needs.
The de novo pathways are almost identical in all organisms.
Salvage pathways
Most organisms have the ability to synthesize nucleotides from
nucleosides or bases that become available through the diet or from
degredation of nucleic acids.
In animals, the extracellular hydrolysis of ingested nucleic acids
represents the major route by which bases become available.
Reutilization and catabolism of purine and pyrimidine bases
blue-catabolism
red-salvage pathways
endonucleases:
pancreatic RNAse
pancreatic DNAse
phosphodiesterases:
usually non-specific
PRPP: a central metabolite in de novo and salvage pathways
PRPP synthetase
Enzyme inhinited by AMP, ADP, and GDP. In E. coli, expression is repressed
by PurR repressor bound to either guanine or hypoxanthine.
Roles of PRPP: his and trp biosynthesis, nucleobase salvage pathways, de
novo synthesis of nucleotides
Purine Nucleotide Synthesis
O
COO
OOC
2-
O3P O CH2
H
O
H

H
H
C
OH
OH
OH
Aspartate
+ ATP
CH
5
ADP
+ Pi
HC
N
H
SAICAR Synthetase
CH2
C
COO
Ribose
Phosphate
Pyrophosphokinase
AIR
Car boxylase
AMP
ADP + Pi
O3P O CH2
O
H
H
OH
OH
H
H

O
Ribose-5-Phosphate
Fumarate
P
O
O
O
P
O
C
O
C
H2N
CH
5
N
C
5-Aminoimidazole Ribotide (AIR)
ADP + Pi
Transferase
O3P O CH2
H
H
OH
OH
HN

C
H2C
O
ADP +
Glutamate + Pi
FGAM
Synthetase
ADP
+ Pi
C
H
NH2
Ribose-5-Phosphate
5-Formaminoimidazole-4-carboxamide
ribotide (FAICAR)
ATP +
Glutamine +
H2O
H 2C
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
O
NH
Formylglycinamidine ribotide (FGAM)
N
C4
Ribose-5-Phosphate
GAR Synthetase
THF
C
H2N
H
-5-Phosphoribosylamine (PRA)
H
CH
O
Glycine
+ ATP
AICAR
Transformylase
O
C
NH2
O
H
O3P O CH2
N10-FormylTHF
H
N
H2C
2-
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
2-
CH
5
H2N
Ribose-5-Phosphate
Carboxyamidoimidazole Ribotide (CAIR)
N
C4
N
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)
Example of a salvage pathway: guanine phosphoribosyl transferase
In vivo, the reaction is driven to the right by the action of pyrophosphatase
Shown: HGPRT, cells also have a APRT.
De novo biosynthesis of purines: low molecular weight
precursors of the purine ring atoms
Synthesis of IMP
The base in IMP is called
hypoxanthine
Note: purine ring built up at
nucleotide level.
precursors:
glutamine (twice)
glycine
N10-formyl-THF (twice)
HCO3
aspartate
In vertebrates, 2,3,5 catalyzed
by trifunctional enzyme,
6,7 catalyzed by bifunctional
enzyme.
Pathways from IMP to AMP and GMP
G-1: IMP dehydrogenase
G-2: XMP aminase
A-1: adenylosuccinate
synthetase
A-2: adenylosuccinate lyase
Note: GTP used to make
AMP, ATP used to make
GMP.
Also, feedback inhibition by
AMP and GMP.
Pathways from AMP and GMP to ATP and GTP
Conversion to diphosphate involves specific kinases:
GMP + ATP <-------> GDP + ADP Guanylate kinase
AMP + ATP <-------> 2 ADP
Adenylate kinase
Conversion to triphosphate by Nucleoside diphosphate kinase (NDK):
GDP + ATP <------> GTP + ADP DG0’= 0
ping pong reaction mechanism with phospho-his intermediate.
NDK also works with pyrimidine nucleotides and is driven by mass action.
Allosteric regulation of purine de novo synthesis
Purine degredation
AMP deamination in muscle, hydrolysis in other tissues.
Xanthine oxidase:contains FAD, molybdenum, and non-heme iron.
In primates, uric acid is the end product, which is excreted.
Purine degredation in
other animals
Clinical disorders of purine metabolism
Excessive accumulation of uric acid: Gout
The three defects shown each result in elevated de novo
purine biosynthesis
Common treatment for gout: allopurinol
Allopurinol is an analogue of hypoxanthine that strongly
inhibits
xanthine oxidase. Xanthine and hypoxanthine, which
are soluble, are accumulated and excreted.
Diseases of purine metabolism (continued)
Lesch-Nyhan Syndrome: Severe HGPRT deficiency
In addition to symptoms of gout, patients display severe behavioral
disorders, learning disorder, aggressiveness and hostility, including selfdirected. Patients must be restrained to prevent self-mutilation. Reason
for the behavioral disorder is unknown.
X-linked trait (HGPRT gene is on X chromosome).
Severe combined immune deficiency (SCID): lack of adenosine
deaminase (ADA).
Lack of ADA causes accumulation of deoxyadenosine.
Immune cells, which have potent salvage pathways, accumulate dATP,
which blocks production of other dNTPs by its action on ribonucleotide
reductase. Immune cells can’t replicate their DNA, and thus can’t
mount an immune response.
De novo pyrimidine biosynthesis
Pyrimidine ring is assembled as the free base, orotic acid, which is
them converted to the nucleotide orotidine monophosphate (OMP).
The pathway is unbranched. UTP is a substrate for formation of
CTP.
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
O
O
C
C
O
CH
N
H
CH
O
2-
CH
N
H
COO
COO
O3P
O
CH2
CH
N
O
H2O
Dihydroorotase
Carbamoyl Aspartate
C
CH2
HN
C
C
C
HN
CH2
NH2
CO2
Quinone
Pi
HO
OMP
Decarboxylase
Dihydroorotate
Dehydrogenase
O
H
H
OH
OH
H

H
Dihydroorotate
Uridine Monophosphate
(UMP)
De novo synthesis of pyrimidines
1: carbamyl phosphate
synthase
2: aspartate
transcarbamylase
3: dihydroorotase
4: dihydroorotate DH
5: orotate
phosphoribosyl
tranferase
6: orotidylate
decarboxylase
7: UMP kinase
8: NDK
9: CTP synthetase
CAD=1,2,3
5 +6=single protein
Regulation of
pyrimidine
de novo synthesis
Catabolism of
pyrimidines
Overview of dNTP biosynthesis
One enzyme, ribonucleotide reductase,
reduces all four ribonucleotides to their
deoxyribo derivitives.
A free radical mechanism is involved
in the ribonucleotide reductase
reaction.
There are three classes of ribonucleotide
reductase enzymes in nature:
Class I: tyrosine radical, uses NDP
Class II: adenosylcobalamin. uses NTPs
(cyanobacteria, some bacteria,
Euglena).
Class III: SAM and Fe-S to generate
radical, uses NTPs.
(anaerobes and fac. anaerobes).
Structure of rNDP reductase (E. coli, ClassI)
Proposed mechanism
for rNDP reductase
Proposed reaction mechanism for ribonucleotide reductase
Sources of reducing power for rNDP reductase
Biological activities of thioredoxin
Regulation of activities of mammalian rNDP reductase
Salvage and de novo pathways to thymine nucleotides
Substrate recvognition by dUTPase
Relationship between thymidylate synthase and enzymes of
tetrahydrofolate metabolism
Catalytic mechanism of thymidylate
synthase
Regeneration of N5, N10-methylenetetrahydrofolate
Biosynthesis of NAD+ and NADP+
Biosynthesis of CoA from pantothenate
Proposed reaction mechanism for FGAM synthetase
The transformylation reactions are catalyzed by a multiprotein complex
components of the complex:
GAR transformylase (3)
AICAR transformylase (9)
serine hydroxymethyl transferase, trifunctional formylmethenylmethylene-THF synthase (activities shown with asterisk)
Proposed catalytic mechanism for OMP decarboxylase
Reactions catalyzed by eukaryotic dihydroorotate dehydrogenase
Nitrogenous Bases
Planar, aromatic, and heterocyclic
Derived from purine or pyrimidine
Numbering of bases is “unprimed”

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Purines
Nitrogenous Bases
N1: Aspartate Amine
C2, C8: Formate
N3, N9: Glutamine
C4, C5, N7: Glycine
C6: Bicarbonate Ion
Pyrimidines
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
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Purine Nucleotides
Get broken down into Uric Acid (a purine)

Purine Nucleotide Synthesis
ATP is involved in 6 steps and an additional ATP is needed to form the
first molecule (R5P)

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 3, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)


Coupling of Reactions
Hydrolyzing a phosphate from ATP is relatively easy
DG°’= -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:
or
Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl,
adenosinyl group in transferase reaction
OR
ATP hydrolysis can drive an otherwise unfavorable reaction
(synthetase; “energase”)

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Purine Biosynthetic Pathway
Coupling 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 is coupled:
Reactions 3, 4, 6
Reactions 7, 8
Reactions 10, 11
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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
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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
At level of IMP production:
Independent control
Synergistic control
Feedforward activation by PRPP
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Below level of IMP production
Reciprocal control

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Total amounts of purine nucleotides controlled
Relative amounts of ATP, GTP controlled

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Purine Catabolism and Salvage
All purine degradation in animals leads to uric acid
Ingested nucleic acids are degraded 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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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
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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
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Transfers electrons to O2  H2O2
H2O2 is toxic
Disproportionated to H2O and O2 by catalase
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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
Purine Nucleotide Cycle
In-Class Question: Why is the purine nucleotide cycle
important in muscle metabolism during a burst of
activity?
Adenosine Deaminase
CHIME Exercise: 2ADA
Enzyme catalyzing deamination of Adenosine to Inosine 
/ barrel domain structure 
“TIM Barrel” – central barrel structure with 8 twisted parallel 
-strands connected by 8 -helical loops
Active site is at bottom of funnel-shaped pocket formed by 
loops
Found in all glycolytic enzymes 
Found in proteins that bind and transport metabolites 
Uric Acid Excretion
Humans – excreted into urine as insoluble
crystals
Birds, terrestrial reptiles, some insects – excrete
isoluble crystals in paste form (conserve water)
Others – further modification :
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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 !
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.
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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.
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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?
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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”
Lesch-Nyhan Syndrome
A defect in production or activity of 
HGPRT
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)
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Causes gout-like symptoms, but also
neurological symptoms  spasticity,
aggressiveness, self-mutilation
First neuropsychiatric abnormality that was
attributed to a single enzyme
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Purine Autism
25% of autistic patients may
overproduce purines
To diagnose, must test urine over 24
hours
Biochemical findings from this test 
disappear in adolescence
Must obtain urine specimen in infancy, 
but it’s difficult to do!
Pink urine due to uric acid crystals may be 
seen in diapers

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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 : Aspartate
C2 : HCO3N3 : Glutamine amide Nitrogen

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
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Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on same
chain

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
O
O
C
C
O
CH
N
H
CH
O
2-
CH
N
H
COO
COO
O3P
O
CH2
CH
N
O
H2O
Dihydroorotase
Carbamoyl Aspartate
C
CH2
HN
C
C
C
HN
CH2
NH2
CO2
Quinone
Pi
HO
OMP
Decarboxylase
Dihydroorotate
Dehydrogenase
O
H
H
OH
OH
H

H
Dihydroorotate
Uridine Monophosphate
(UMP)
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 


OMP DECARBOXYLASE : THE MOST
CATALYTICALLY PROFICIENT ENZYME
FINAL REACTION OF PYRIMIDINE PATHWAY
ANOTHER MECHANISM FOR DECARBOXYLATION
A CARBANION INTERMEDIATE (UNSTABLE)
MUST BE STABILIZED
BUT NO COFACTORS ARE NEEDED!
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SOME OF THE BINDING ENERGY BETWEEN OMP
AND THE ACTIVE SITE IS USED TO STABILIZE THE
TRANSITION STATE
“PREFERENTIAL TRANSITION STATE BINDING”
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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
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*Purine synthesis inhibited by ADP and GDP at ribose
phosphate pyrophosphokinase step, controlling level of
PRPP  also regulates pyrimidines
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
OROTICACIDURIA?
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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 -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 ribonucleotides

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 in vitro
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RIBONUCLEOTIDE REDUCTASE
R1 SUBUNIT
Specificity Site
Hexamerization site
Activity Site
Five redox-active –SH groups from cysteines
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R2 SUBUNIT
Tyr 122 radical
Binuclear Fe(III) complex
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Chime Exercise
E. coli Ribonucleotide Reductase:
3R1R and 4R1R: R1 subunit
1RIB and 1AV8: R2 subunit
Ribonucleotide Reductase R2
Subunit
Fe prosthetic group– binuclear, with each Fe
octahedrally coordinated
Fe’s are bridged by O-2 and carboxyl gp of Glu 115
Tyr 122 is close to the Fe(III) complex  stabilization of a
tyrosyl free-radical
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During the overall process, a pair of –SH groups
provide the reducing equivalents
A protein disulfide group is formed
Gets reduced by two other sulfhydryl gps of Cys residues in
R1
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Mechanism of Ribonucleotide Reductase
Reaction
Free Radical
Involvement of multiple –SH groups
RR is left with a disulfide group that must be
reduced to return to the original enzyme
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RIBONUCLEOTIDE REDUCTASE
ACTIVITY IS RESPONSIVE TO LEVEL OF CELLULAR
NUCLEOTIDES:
ATP ACTIVATES REDUCTION OF
CDP
UDP
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dTTP
INDUCES GDP REDUCTION
INHIBITS REDUCTION OF CDP. UDP
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dATP INHIBITS REDUCTION OF ALL NUCLEOTIDES
dGTP
STIMULATES ADP REDUCTION
INHIBITS CDP,UDP,GDP REDUCTION
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RIBONUCLEOTIDE REDUCTASE
CATALYTIC ACTIVITY VARIES WITH STATE OF
OLIGOMERIZATION:
WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE OF
R1 (CATALYTICALLY INACTIVE MONOMER)
 CATALYTICALLY ACTIVE (R1)2
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WHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERS
(R1)4a
 TETRAMER FORMATION
(ACTIVE STATE) == (R1)4b (INACTIVE)
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WHEN ATP BINDS TO HEXAMERIZATION SITE
 CATALYTICALLY ACTIVE HEXAMERS (R1)6
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Thioredoxin
Physiologic reducing agent of RNR
Cys pair can swap H atoms with disulfide formed
regenerate original enzyme
Thioredoxin gets oxidized to disulfide
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Oxidized Thioredoxin gets reduced by thioredoxin reductase mediated
by NADPH (final electron acceptor)
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Thymine Formation
Formed by methylating deoxyuridine monophosphate
(dUMP)
UTP needed for RNA production, but dUTP not
needed for DNA
If dUTP produced excessively, would cause substitution
errors (dUTP for dTTP)
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dUTP hydrolyzed by dUTP diphosphohydrolase to
dUMP  methylated at C5 to form dTMP
rephosphorylate to form dTTP
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Chime Exercise
1DUD: dUTPase
Tetrahydrofolate (THF)
Methylation of dUMP catalyzed by thymidylate
synthase
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Cofactor: N5,N10-methylene THF 
Oxidized to dihydrofolate 
Only known rxn where net oxidation state of THF
changes
THF Regeneration:
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DHF + NADPH + H+  THF + NADP+ (enzyme: dihydrofolate reductase)
THF + Serine  N5,N10-methylene-THF + Glycine
(enzyme: serine hydroxymethyl transferase)
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)
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Dihydrofolate reductase step can be stopped
competitively (DHF analogs)
Anti-Folates: Aminopterin, methotrexate, trimethoprim
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IN-CLASS QUESTION
IN von GIERKE’S DISEASE, OVERPRODUCTION OF URIC ACID OCCURS. THIS
DISEASE IS CAUSED BY A DEFICIENCY OF
GLUCOSE-6-PHOSPHATASE.
EXPLAIN THE BIOCHEMICAL EVENTS THAT LEAD
TO INCREASED URIC ACID PRODUCTION?
WHY DOES HYPOGLYCEMIA OCCUR IN THIS
DISEASE?
WHY IS THE LIVER ENLARGED?
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ADENOSINE DEAMINASE DEFICIENCY
IN PURINE DEGRADATION, ADENOSINE  INOSINE
ENZYME IS ADA
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ADA DEFICIENCY RESULTS IN SCID
“SEVERE COMBINED IMMUNODEFICIENCY”
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SELECTIVELY KILLS LYMPHOCYTES
BOTH B- AND T-CELLS
MEDIATE MUCH OF IMMUNE RESPONSE
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ALL KNOWN ADA MUTANTS STRUCTURALLY
PERTURB ACTIVE SITE
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ADA DEFICIENCY
IN-CLASS QUESTION: EXPLAIN THE BIOCHEMISTRY
THAT RESULTS WHEN A PERSON HAS ADA
DEFICIENCY
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(HINT: LYMPHOID TISSUE IS VERY ACTIVE IN
DEOXYADENOSINE PHOSPHORYLATION)
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ADA DEFICIENCY
ONE OF FIRST DISEASES TO BE TREATED WITH
GENE THERAPY
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ADA GENE INSERTED INTO LYMPHOCYTES; THEN
LYMPHOCYTES RETURNED TO PATIENT
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PEG-ADA TREATMENTS
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ACTIVITY LASTS 1-2 WEEKS
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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
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
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Nucleosides
Phosphate Groups
Mono-, di- or triphosphates
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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 Thymine 
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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”
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Adenosine, Cytosine 
Pyrimidine nucleosides end in “-dine” 
Thymidine, Guanidine 
Nucleotides:
Start with the nucleoside name from above and add 
“mono-”, “di-”, or “triphosphate”
Adenosine Monophosphate, Guanidine Triphosphate, 
Deoxythymidine Diphosphate
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In-Class Activities
Look at the Nucleotide Structures
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Take the Nucleotide Identification Quiz
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Be prepared to identify some of these structures
on an exam. Learn some “tricks” that help you to
distinguish among the different structures
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