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Nucleotides: Synthesis and
Degradation
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Nitrogenous Bases
Planar, aromatic, and heterocyclic
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 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”
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
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In-Class Activities
Look at the Nucleotide Structures
Take the Nucleotide Identification Quiz
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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Nucleotide MetabolismPURINE 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)
Buchanan (mid 1900s) showed where purine
ring components came from:
N1: Aspartate AmineC2, C8: Formate
N3, N9: GlutamineC4, C5, N7: Glycine
C6: Bicarbonate Ion
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Purine Nucleotide Synthesis
OH
H
H
CH2
OH OH
H HO
α
O2-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
β
O2-O3P
β-5-Phosphoribosylamine (PRA)
Amido phosphoribosyl
Transf erase
Glutamine + H2O
Glutamate
+ PPi
H
NH
H
CH2
OH OH
H HO
O2-O3P
CO
H2C NH2
Glycinamide Riboti de (GAR)
GAR Syn thetase
Glycine + ATP
ADP
+ Pi
H2C
C
NH
O
CH
HN
O
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
H2C
C
NH
O
CH
HN
HN
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
THFN10-Formyl-THF
GAR Transformylase
ATP +
Glutamine +
H 2O
ADP +
Glutamate + PiFGAM
Synthetase
HC
CN
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole Ribo tide (AIR)
ATP
ADP + PiAIR
Synthetase
C
CN
CH
N
H2N
OOC
Ribose-5-Phosphate
4
5
Carboxyamidoimidazole Ribotide (CAIR)
ATP
+HCO3
ADP + PiAIR Car boxylase
Aspartate
+ ATPADP
+ Pi
SAICAR Synthetase
Adenylosuccinate
Lyase
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-carboxamide
ribotide (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
HC
N
C
C
C
N
CH
N
O
4
5
HH
CH2
OH OH
H HO
O2-O3P
IMP
Cyclohydrolase
H2O
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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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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”)
Coupling of Reactions
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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, 11
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In Class Activity
***
Calculate how many ATP equivalents are needed for the de novo synthesize IMP. Assume that all of the substrates (R5P, glutamine, etc) are available
Note: You should be able to do this calculation for the synthesis of
any of the nucleoside monophosphates
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IMP Conversion to AMP
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IMP Conversion to GMP
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Regulatory Control of Purine
Nucleotide BiosynthesisGTP 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
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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
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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 intestineGroup-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
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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Adenosine Degradation
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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
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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
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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
THE PURINE NUCLEOTIDE CYCLE
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Purine Nucleotide Cycle
***
In-Class Question: Why is the purine nucleotide
cycle important in muscle metabolism during a
burst of activity?
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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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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?
� 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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Choi HK, Atkinson K, Karlson EW et al. . 2004. “Alcohol intake and risk of incident gout in men:
a prospective study”. Lancet 363: 1277-1281
ALCOHOL CONSUMPTION AND GOUT
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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)
� 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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IN-CLASS QUESTION
***
� IN von GIERKE’S DISEASE, OVERPRO-DUCTION 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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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 : HCO3-
N3 : Glutamine amide Nitrogen
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2 ATP + HCO3- + Glutamine + H2O
CO
O PO3-2
NH2
Carbamoyl Phosphate
NH2
C
N
H
CH
CH2
C
COOO
HO
O
Carbamoyl Aspartate
HN
C
N
H
CH
CH2
C
COOO
O
Dihydroorotate
HN
C
N
H
C
CH
C
COOO
O
Orotate
HN
C
N
C
CH
C
COOO
O
HH
CH2
OH OH
H H
OO2-O3P
β
Orotidine-5'-monophosphate
(OMP)
HN
C
N
CH
CH
C
O
O
HH
CH2
OH OH
H H
OO
2-O3P
β
Uridine Monophosphate
(UMP)
2 ADP +
Glutamate +
Pi
CarbamoylPhosphateSynthetase II
Aspartate
Transcarbamoylase
(ATCase)
Aspartate
Pi
H2O
Dihydroorotase
Quinone
Reduced
QuinoneDihydroorotateDehydrogenase
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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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”
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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 Synthetasedriven 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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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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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
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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
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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• Three allosteric sites
� Specificity Site
� Hexamerization site
� Activity Site
• Five redox-active –SH groups from cysteines
� R2 SUBUNIT• Tyr 122 radical
• Binuclear Fe(III) complex
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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
� During the overall process, a pair of –SH groups provides 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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Chime Exercise
E. coli Ribonucleotide Reductase:
3R1R and 4R1R: R1 subunit1RIB and 1AV8: R2 subunit
• Explore 1AV8: Ribonucleotide Reductase in detail.This is the R2 subunit of E. coli Ribonucleotide Reductase. The biological molecule consists of a heterotetramer of 2 R1 and two R2 chains.
• Identify the following structures:
– 8 long αααα -helices in one unit of R2– Tyr 122 residue
– The binuclear Fe (III) complex
– The ligands of the Fe (III) complex
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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
• dTTP
� INDUCES GDP REDUCTION
� INHIBITS REDUCTION OF CDP. UDP
• 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
• WHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERS
� � TETRAMER FORMATION
� (R1)4a (ACTIVE STATE) == (R1)4b (INACTIVE)
• 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
Oxidized Thioredoxin gets reduced by NADPH ( final electron acceptor)mediated by thioredoxin reductase
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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
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CHIME EXERCISE: dUTPase
� 1DUD: Deoxyuridine-5'-Nucleotide Hydrolase in a complex with a bound substrate analog, Deoxyuridine-5'-Diphosphate (dUDP).
� Explore dUTPase as follows:
• Find the substrate in its binding site
• Find C5 on the Uracil group. Is there enough room to attach a methyl group to C5?
• Locate the ribose 2’ C. What protein group sterically prevents an –OH group from being attached to the 2’ C atom?
• Find the H-bond donors and acceptors (to the uracil base) from the protein. What would be the effect on the H-bonding if the base was changed to cytosine?
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Tetrahydrofolate (THF)
� Methylation of dUMP catalyzed by thymidylate synthase • Cofactor: N5,N10-methylene THF
� Oxidized to dihydrofolate
� Only known rxn where net oxidation state of THF changes
� THF Regeneration:
DHF + NADPH + H+ � THF + NADP+ (enzyme: dihydrofolate
reductase)
THF + Serine � N5,N10-methylene-THF + Glycine
(enzyme: serine hydroxymethyl transferase)
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dUMP dTMP
NADPH + H+
NADP+
SERINE
GLYCINE
REGENERATION OF N5,N10 METHYLENETETRAHYDROFOLATE
DHFN5,N10 – METHYLENE-THF
THF
dihydrofolate reductaseserine hydroxymethyl
transferase
thymidylate synthase
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dUMP dTMP
NADPH + H+
NADP+
SERINE
GLYCINE
INHIBITORS OF N5,N10 METHYLENETETRAHYDROFOLATE
REGENERATION
DHFN5,N10 – METHYLENE-THF
THF
dihydrofolate reductaseserine hydroxymethyl
transferase
thymidylate synthase
METHOTREXATEAMINOPTERINTRIMETHOPRIM
FdUMP
X
X
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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
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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
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Adenosine Deaminase
CHIME Exercise: 2ADA
Enzyme catalyzing deamination of Adenosine to Inosine
α/β barrel domain structure
– “TIM Barrel” – central barrel structure with 8 twisted
parallel β-strands connected by 8 α-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
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ADA DEFICIENCY
***
� IN-CLASS QUESTION: EXPLAIN THE BIOCHEMISTRY THAT RESULTS WHEN A PERSON HAS ADA DEFICIENCY
� (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
� ADA GENE INSERTED INTO LYMPHOCYTES; THEN LYMPHOCYTES RETURNED TO PATIENT
� PEG-ADA TREATMENTS• ACTIVITY LASTS 1-2 WEEKS