Nucleotides_revised published by the way

Nuclear

1. Nucleotides:Nucleotides: Synthesis and Synthesis and
DegradationDegradation

2. Nitrogenous BasesNitrogenous Bases
Planar, aromatic, and heterocyclicPlanar, aromatic, and heterocyclic
Derived from Derived from purinepurine or or pyrimidinepyrimidine
Numbering of bases is “unprimed”Numbering of bases is “unprimed”

3. Nucleic Acid BasesNucleic Acid Bases
Purines
Pyrimidines

4. SugarsSugars
Pentoses (5-C sugars)Pentoses (5-C sugars)
Numbering of sugars is “primed”Numbering of sugars is “primed”

5. SugarsSugars
D-Ribose and 2’-DeoxyriboseD-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group

6. NucleosidesNucleosides
Result from linking one of the sugars with Result from linking one of the sugars with
a purine or pyrimidine base through an N-a purine or pyrimidine base through an N-
glycosidic linkageglycosidic linkage
–Purines bond to the C1’ carbon of the sugar at Purines bond to the C1’ carbon of the sugar at
their N9 atomstheir N9 atoms
–Pyrimidines bond to the C1’ carbon of the Pyrimidines bond to the C1’ carbon of the
sugar at their N1 atomssugar at their N1 atoms

7. NucleosidesNucleosides

8. Phosphate GroupsPhosphate Groups
Mono-, di- or triphosphatesMono-, di- or triphosphates
Phosphates can be bonded to either C3 or Phosphates can be bonded to either C3 or
C5 atoms of the sugarC5 atoms of the sugar

9. NucleotidesNucleotides
Result from linking one or more phosphates Result from linking one or more phosphates
with a nucleoside onto the 5’ end of the with a nucleoside onto the 5’ end of the
molecule through esterificationmolecule through esterification

10. NucleotidesNucleotides
RNA (ribonucleic acid) is a polymer of RNA (ribonucleic acid) is a polymer of
ribonucleotidesribonucleotides
DNA (deoxyribonucleic acid) is a polymer DNA (deoxyribonucleic acid) is a polymer
of deoxyribonucleotidesof deoxyribonucleotides
Both deoxy- and ribonucleotides contain Both deoxy- and ribonucleotides contain
Adenine, Guanine and CytosineAdenine, Guanine and Cytosine
–Ribonucleotides contain UracilRibonucleotides contain Uracil
–Deoxyribonucleotides contain ThymineDeoxyribonucleotides contain Thymine

11. NucleotidesNucleotides
Monomers for nucleic acid polymersMonomers for nucleic acid polymers
Nucleoside Triphosphates are important Nucleoside Triphosphates are important
energy carriers (ATP, GTP)energy carriers (ATP, GTP)
Important components of coenzymesImportant components of coenzymes
–FAD, NADFAD, NAD
++
and Coenzyme A and Coenzyme A

12. Naming ConventionsNaming Conventions
Nucleosides:Nucleosides:
–Purine nucleosides end in “-sine” Purine nucleosides end in “-sine”
Adenosine, GuanosineAdenosine, Guanosine
–Pyrimidine nucleosides end in “-dine”Pyrimidine nucleosides end in “-dine”
Thymidine, Cytidine, UridineThymidine, Cytidine, Uridine
Nucleotides:Nucleotides:
–Start with the nucleoside name from above Start with the nucleoside name from above
and add “mono-”, “di-”, or “triphosphate”and add “mono-”, “di-”, or “triphosphate”
Adenosine Monophosphate, Cytidine Triphosphate, Adenosine Monophosphate, Cytidine Triphosphate,
Deoxythymidine DiphosphateDeoxythymidine Diphosphate

13. In-Class ActivitiesIn-Class Activities
Look at the Look at the Nucleotide StructuresNucleotide Structures
Take the Take the Nucleotide Identification QuizNucleotide Identification Quiz
Be prepared to identify some of these Be prepared to identify some of these
structures on an exam. Learn some structures on an exam. Learn some
“tricks” that help you to distinguish among “tricks” that help you to distinguish among
the different structuresthe different structures

14. Nucleotide MetabolismNucleotide Metabolism
PURINE RIBONUCLEOTIDES: formed PURINE RIBONUCLEOTIDES: formed de novode novo
–i.e., purines are i.e., purines are notnot initially synthesized as free bases initially synthesized as free bases
–First purine derivative formed is Inosine Mono-First purine derivative formed is Inosine Mono-
phosphate (IMP)phosphate (IMP)
The purine base is The purine base is hypoxanthinehypoxanthine
AMP and GMP are formed from IMPAMP and GMP are formed from IMP

15. Purine NucleotidesPurine Nucleotides
Get broken down into Uric Acid (a purine) Get broken down into Uric Acid (a purine)
Buchanan (mid 1900s) showed where purine Buchanan (mid 1900s) showed where purine
ring components came from:ring components came from:
N
1
: Aspartate Amine
C2, C8: Formate
N3, N9: Glutamine
C4, C5, N7: Glycine
C6: Bicarbonate Ion

16. Purine Nucleotide Synthesis
OH
H
H
CH
2
OH OH
H H
O

O
2-
O
3P
-D-Ribose-5-Phosphate (R5P)
O
H
H
CH
2
OH OH
H H
O

O
2-
O
3P
5-Phosphoribosyl--pyrophosphate (PRPP)
P
O
O
OP
O
O
O
ATP
AMP
Ribose
Phosphate
Pyrophosphokinase
H
NH
2
H
CH
2
OH OH
H H
O

O
2-
O
3P
-5-Phosphoribosylamine (PRA)
Amidophosphoribosyl
Transferase
Glutamine
+ H
2O
Glutamate
+ PP
i
H
NH
H
CH
2
OH OH
H H
O
O
2-
O
3P
CO
H
2CNH
2
Glycinamide Ribotide (GAR)
GAR Synthetase
Glycine
+ ATP
ADP
+ P
i
H
2C
C
NH
O
CH
H
N
O
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
H
2C
C
NH
O
CH
H
N
HN
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
THFN
10
-Formyl-THF
GAR Transformylase
ATP +
Glutamine +
H
2O
ADP +
Glutamate + P
i
FGAM
Synthetase
HC
C
N
CH
N
H
2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole Ribotide (AIR)
ATP
ADP + P
i
AIR
Synthetase
C
C
N
CH
N
H
2N
OOC
Ribose-5-Phosphate
4
5
Carboxyamidoimidazole Ribotide (CAIR)
ATP
+HCO
3
ADP + P
i
AIR
Car boxylase
Aspartate
+ ATP
ADP
+ P
i
SAICAR Synthetase
Adenylosuccinate
Lyase
Fumarate
C
C
N
CH
N
NH
Ribose-5-Phosphate
4
5
5-Formaminoimidazole-4-carboxamide
ribotide (FAICAR)
C
H
2N
O
C
H
O
C
C
N
CH
N
H
2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-carboxamide
ribotide (AICAR)
C
H
2N
O
C
C
N
CH
N
H
2N
C
N
H
O
HC
COO
CH
2
COO
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-(N-succinylocarboxamide)
ribotide (SAICAR)
THF
AICAR
Transformylase
N
10
-Formyl-
THF
Inosine Monophosphate (IMP)
HN
HC
N
C
C
C
N
CH
N
O
4
5
HH
CH2
OH OH
H H
O
O
2-
O
3P
IMP
Cyclohydrolase
H
2O

17. Purine Nucleotide Synthesis Purine Nucleotide Synthesis
at a Glanceat a Glance
ATP is involved in 6 stepsATP is involved in 6 steps
PRPP in the first step of Purine synthesis is also a precursor for PRPP in the first step of Purine synthesis is also a precursor for
Pyrimidine Synthesis, His and Trp synthesisPyrimidine Synthesis, His and Trp synthesis
–Role of ATP in first step is unique– group transfer rather than Role of ATP in first step is unique– group transfer rather than
couplingcoupling
In second step, CIn second step, C11 notation changes from notation changes from  to to  (anomers (anomers
specifying OH positioning on Cspecifying OH positioning on C11 with respect to C with respect to C44 group) group)
In step 2, PPIn step 2, PPii is hydrolyzed to 2P is hydrolyzed to 2Pii (irreversible, “committing” step) (irreversible, “committing” step)

18. Hydrolyzing a phosphate from ATP is relatively easy Hydrolyzing a phosphate from ATP is relatively easy
G°’= -30.5 kJ/molG°’= -30.5 kJ/mol
–If endergonic reaction released energy into cell as heat energy, If endergonic reaction released energy into cell as heat energy,
wouldn’t be useful wouldn’t be useful
–Must be coupled to an exergonic reactionMust be coupled to an exergonic reaction
When ATP is a reactant:When ATP is a reactant:
–Part of the ATP can be transferred to an acceptor: PPart of the ATP can be transferred to an acceptor: Pii, PP, PPii, adenyl, , adenyl,
or adenosinyl group or adenosinyl group
–ATP hydrolysis can drive an otherwise unfavorable reactionATP hydrolysis can drive an otherwise unfavorable reaction
(synthetase; “energase”)(synthetase; “energase”)
Coupling of ReactionsCoupling of Reactions

19. Purine Biosynthetic PathwayPurine Biosynthetic Pathway
Channeling of some reactions on pathway organizes and Channeling of some reactions on pathway organizes and
controls processing of substrates to products in each stepcontrols processing of substrates to products in each step
–Increases overall rate of pathway and protects intermediates from Increases overall rate of pathway and protects intermediates from
degradationdegradation
In animals, IMP synthesis pathway shows channeling at:In animals, IMP synthesis pathway shows channeling at:
–Reactions 3, 4, 6Reactions 3, 4, 6
–Reactions 7, 8Reactions 7, 8
–Reactions 10, 11Reactions 10, 11

20. In Class ActivityIn Class Activity
******
Calculate how many ATP equivalents are needed for the Calculate how many ATP equivalents are needed for the de novode novo synthesize IMP. synthesize IMP.
Assume that all of the substrates (R5P, glutamine, etc) are availableAssume that all of the substrates (R5P, glutamine, etc) are available
Note: You should be able to do this calculation for the synthesis of Note: You should be able to do this calculation for the synthesis of
any of the nucleoside monophosphatesany of the nucleoside monophosphates

21. IMP Conversion to AMP IMP Conversion to AMP

22. IMP Conversion to GMPIMP Conversion to GMP

23. Regulatory Control of Purine Regulatory Control of Purine
Nucleotide BiosynthesisNucleotide Biosynthesis
GTP is involved in AMP synthesis and ATP is involved in GTP is involved in AMP synthesis and ATP is involved in
GMP synthesis (reciprocal control of production)GMP synthesis (reciprocal control of production)
PRPP is a biosynthetically “central” molecule (why?)PRPP is a biosynthetically “central” molecule (why?)
–ADP/GDP levels – negative feedback on Ribose Phosphate ADP/GDP levels – negative feedback on Ribose Phosphate
Pyrophosphokinase Pyrophosphokinase
–Amidophosphoribosyl transferase is activated by PRPP levelsAmidophosphoribosyl transferase is activated by PRPP levels
–APRT activity has negative feedback at two sitesAPRT activity has negative feedback at two sites
ATP, ADP, AMP bound at one siteATP, ADP, AMP bound at one site
GTP,GDP AND GMP bound at the other siteGTP,GDP AND GMP bound at the other site
Rate of AMP production increases with increasing Rate of AMP production increases with increasing
concentrations of GTP; rate of GMP production concentrations of GTP; rate of GMP production
increases with increasing concentrations of ATPincreases with increasing concentrations of ATP

24. Regulatory Control of Purine BiosynthesisRegulatory Control of Purine Biosynthesis
Above the level of IMP production:Above the level of IMP production:
–Independent controlIndependent control
–Synergistic controlSynergistic control
–Feedforward activation by PRPPFeedforward activation by PRPP
Below level of IMP productionBelow level of IMP production
–Reciprocal controlReciprocal control
Total amounts of purine nucleotides controlledTotal amounts of purine nucleotides controlled
Relative amounts of ATP, GTP controlledRelative amounts of ATP, GTP controlled

25. Purine Catabolism and SalvagePurine Catabolism and Salvage
All purine degradation leads to All purine degradation leads to uric acid uric acid (but it might not (but it might not
stop there)stop there)
Ingested nucleic acids are degraded to nucleotides by Ingested nucleic acids are degraded to nucleotides by
pancreatic nucleases, and intestinal phosphodiesterases pancreatic nucleases, and intestinal phosphodiesterases
in the intestinein the intestine
Group-specific nucleotidases and non-specific Group-specific nucleotidases and non-specific
phosphatases degrade nucleotides into nucleosidesphosphatases degrade nucleotides into nucleosides
–Direct absorption of nucleosides Direct absorption of nucleosides
–Further degradation Further degradation
Nucleoside + HNucleoside + H22O O  base + ribose (nucleosidase) base + ribose (nucleosidase)
Nucleoside + PNucleoside + P
ii  base + r-1-phosphate (n. phosphorylase) base + r-1-phosphate (n. phosphorylase)
NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND
EXCRETED.EXCRETED.

26. Intracellular Purine CatabolismIntracellular Purine Catabolism
Nucleotides broken into nucleosides by action of Nucleotides broken into nucleosides by action of
5’-nucleotidase (hydrolysis reactions)5’-nucleotidase (hydrolysis reactions)
Purine nucleoside phosphorylase (PNP)Purine nucleoside phosphorylase (PNP)
–Inosine Inosine  Hypoxanthine Hypoxanthine
–Xanthosine Xanthosine  Xanthine Xanthine
–Guanosine Guanosine  Guanine Guanine
–Ribose-1-phosphate splits offRibose-1-phosphate splits off
Can be isomerized to ribose-5-phosphateCan be isomerized to ribose-5-phosphate
Adenosine is deaminated to Inosine (ADA)Adenosine is deaminated to Inosine (ADA)

27. Intracellular Purine CatabolismIntracellular Purine Catabolism
Xanthine is the point of convergence for the Xanthine is the point of convergence for the
metabolism of the purine basesmetabolism of the purine bases
Xanthine Xanthine  Uric acid Uric acid
–Xanthine oxidase catalyzes two reactionsXanthine oxidase catalyzes two reactions
Purine ribonucleotide degradation pathway Purine ribonucleotide degradation pathway
is same for purine deoxyribonucleotidesis same for purine deoxyribonucleotides

28. Adenosine Degradation

29. Xanthosine DegradationXanthosine 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

30. Xanthine Oxidase Xanthine Oxidase
A homodimeric proteinA homodimeric protein
Contains electron transfer proteinsContains electron transfer proteins
– FADFAD
–Mo-pterin complex in +4 or +6 stateMo-pterin complex in +4 or +6 state
– Two 2Fe-2S clustersTwo 2Fe-2S clusters
Transfers electrons to OTransfers electrons to O22  H H
22OO
22
– HH22OO22 is toxic is toxic
– Disproportionated to HDisproportionated to H22O and OO and O22 by catalase by catalase

31. AMP + HAMP + H22O O  IMP + NH IMP + NH44
++
(AMP Deaminase)(AMP Deaminase)
IMP + Aspartate + GTP IMP + Aspartate + GTP  AMP + Fumarate + GDP + P AMP + Fumarate + GDP + Pii
(Adenylosuccinate Synthetase)(Adenylosuccinate Synthetase)
COMBINE THE TWO REACTIONS:COMBINE THE TWO REACTIONS:
Aspartate + HAspartate + H22O + GTP O + GTP  Fumarate + GDP + P Fumarate + GDP + Pii + + NHNH44
++
The overall result of combining reactions is deamination of Aspartate to The overall result of combining reactions is deamination of Aspartate to
Fumarate at the expense of a GTP Fumarate at the expense of a GTP
THE PURINE NUCLEOTIDE CYCLETHE PURINE NUCLEOTIDE CYCLE

32. Purine Nucleotide CyclePurine Nucleotide Cycle
******
In-Class Question: Why is the purine nucleotide In-Class Question: Why is the purine nucleotide
cycle important in muscle metabolism during a cycle important in muscle metabolism during a
burst of activity?burst of activity?

33. Uric Acid ExcretionUric Acid Excretion
Humans – excreted into urine as insoluble Humans – excreted into urine as insoluble
crystalscrystals
Birds, terrestrial reptiles, some insects – Birds, terrestrial reptiles, some insects –
excrete insoluble crystals in paste form excrete insoluble crystals in paste form
–Excess amino N converted to uric acidExcess amino N converted to uric acid
(conserves water)(conserves water)
Others – further modification :Others – further modification :
Uric Acid Uric Acid  Allantoin Allantoin  Allantoic Acid Allantoic Acid  Urea Urea  Ammonia Ammonia

34. Purine Purine SalvageSalvage
Adenine phosphoribosyl transferase (APRT)Adenine phosphoribosyl transferase (APRT)
Adenine + PRPP Adenine + PRPP  AMP + PP AMP + PPii
Hypoxanthine-Guanine phosphoribosyl transferase Hypoxanthine-Guanine phosphoribosyl transferase
(HGPRT)(HGPRT)
Hypoxanthine + PRPP Hypoxanthine + PRPP  IMP + PP IMP + PPii
Guanine + PRPP Guanine + PRPP  GMP + PP GMP + PPii
(NOTE: THESE ARE ALL (NOTE: THESE ARE ALL REVERSIBLEREVERSIBLE REACTIONS) REACTIONS)
AMP,IMP,GMP do not need to be resynthesized AMP,IMP,GMP do not need to be resynthesized
de novo !de novo !

35. A CASE STUDY : GOUTA CASE STUDY : GOUT
A 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A PAINFUL A 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A PAINFUL
AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS NIGHT AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS NIGHT
HE HAD EATEN A MEAL OF FRIED LIVER AND ONIONS, AFTER HE HAD EATEN A MEAL OF FRIED LIVER AND ONIONS, AFTER
WHICH HE MET WITH HIS POKER GROUP AND DRANK A WHICH HE MET WITH HIS POKER GROUP AND DRANK A
NUMBER OF BEERS.NUMBER OF BEERS.
HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS” HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS”
WAS DIAGNOSED, AND SOME TESTS WERE ORDERED. HIS WAS DIAGNOSED, AND SOME TESTS WERE ORDERED. HIS
SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL < SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL <
7.0 mg/dL).7.0 mg/dL).
THE MAN RECALLED THAT HIS FATHER AND HIS THE MAN RECALLED THAT HIS FATHER AND HIS
GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS, OFTEN GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS, OFTEN
COMPLAINED OF JOINT PAIN AND SWELLING IN THEIR FEET.COMPLAINED OF JOINT PAIN AND SWELLING IN THEIR FEET.

36. A CASE STUDY : GOUTA CASE STUDY : GOUT
THE DOCTOR RECOMMENDED THAT THE MAN USE THE DOCTOR RECOMMENDED THAT THE MAN USE
NSAIDS FOR PAIN AND SWELLING, INCREASE HIS NSAIDS FOR PAIN AND SWELLING, INCREASE HIS
FLUID INTAKE (BUT NOT WITH ALCOHOL) AND REST FLUID INTAKE (BUT NOT WITH ALCOHOL) AND REST
AND ELEVATE HIS FOOT. HE ALSO PRESCRIBED AND ELEVATE HIS FOOT. HE ALSO PRESCRIBED
ALLOPURINOL. ALLOPURINOL.
A FEW DAYS LATER THE CONDITION HAD A FEW DAYS LATER THE CONDITION HAD
RESOLVED AND ALLOPURINOL HAD BEEN RESOLVED AND ALLOPURINOL HAD BEEN
STOPPED. A REPEAT URIC ACID LEVEL WAS STOPPED. A REPEAT URIC ACID LEVEL WAS
OBTAINED (7.1 mg/dL). THE DOCTOR GAVE THE OBTAINED (7.1 mg/dL). THE DOCTOR GAVE THE
MAN SOME ADVICE REGARDING LIFE STYLE MAN SOME ADVICE REGARDING LIFE STYLE
CHANGES.CHANGES.

37. GoutGout

Impaired excretion or overproduction of uric Impaired excretion or overproduction of uric
acidacid

Uric acid crystals precipitate into joints Uric acid crystals precipitate into joints
(Gouty Arthritis), kidneys, ureters (stones)(Gouty Arthritis), kidneys, ureters (stones)

Lead impairs uric acid excretion – lead Lead impairs uric acid excretion – lead
poisoning from pewter drinking gobletspoisoning from pewter drinking goblets

Fall of Roman Empire?Fall of Roman Empire?

Xanthine oxidase inhibitors inhibit Xanthine oxidase inhibitors inhibit
production of uric acid, and treat goutproduction of uric acid, and treat gout

Allopurinol treatment – hypoxanthine Allopurinol treatment – hypoxanthine
analog that binds to Xanthine Oxidase to analog that binds to Xanthine Oxidase to
decrease uric acid productiondecrease uric acid production

41. ALLOPURINOL IS A XANTHINE OXIDASE ALLOPURINOL IS A XANTHINE OXIDASE
INHIBITORINHIBITOR
A SUBSTRATE ANALOG IS CONVERTED TO AN A SUBSTRATE ANALOG IS CONVERTED TO AN
INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”

42. 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

43. Lesch-Nyhan SyndromeLesch-Nyhan Syndrome
A defect in production or activity ofA defect in production or activity of
HGPRT HGPRT

Causes increased level of Hypoxanthine and Causes increased level of Hypoxanthine and
Guanine (Guanine ( in degradation to uric acid) in degradation to uric acid)
Also,PRPP accumulatesAlso,PRPP accumulates

stimulates production of purine nucleotides stimulates production of purine nucleotides
(and thereby increases their degradation)(and thereby increases their degradation)
Causes gout-like symptoms, but also Causes gout-like symptoms, but also
neurological symptoms neurological symptoms  spasticity, spasticity,
aggressiveness, self-mutilationaggressiveness, self-mutilation
First neuropsychiatric abnormality that First neuropsychiatric abnormality that
was attributed to a single enzymewas attributed to a single enzyme

44. Purine AutismPurine Autism

25% of autistic patients may 25% of autistic patients may
overproduce purinesoverproduce purines

To diagnose, must test urine over To diagnose, must test urine over
24 hours24 hours

Biochemical findings from this test Biochemical findings from this test
disappear in adolescencedisappear in adolescence

Must obtain urine specimen in Must obtain urine specimen in
infancy, but it’s difficult to do!infancy, but it’s difficult to do!
•Pink urine due to uric acid crystals may Pink urine due to uric acid crystals may
be seen in diapersbe seen in diapers

45. IN-CLASS QUESTIONIN-CLASS QUESTION
******

IN von GIERKE’S DISEASE, OVERPRO- IN von GIERKE’S DISEASE, OVERPRO-
DUCTION OF URIC ACID OCCURS. THIS DUCTION OF URIC ACID OCCURS. THIS
DISEASE IS CAUSED BY A DEFICIENCY OF DISEASE IS CAUSED BY A DEFICIENCY OF
GLUCOSE-6-PHOSPHATASE.GLUCOSE-6-PHOSPHATASE.
•EXPLAIN THE BIOCHEMICAL EVENTS THAT EXPLAIN THE BIOCHEMICAL EVENTS THAT
LEAD TO INCREASED URIC ACID LEAD TO INCREASED URIC ACID
PRODUCTION?PRODUCTION?
•WHY DOES HYPOGLYCEMIA OCCUR IN THIS WHY DOES HYPOGLYCEMIA OCCUR IN THIS
DISEASE?DISEASE?
•WHY IS THE LIVER ENLARGED?WHY IS THE LIVER ENLARGED?

46. Pyrimidine Ribonucleotide Pyrimidine Ribonucleotide
SynthesisSynthesis

Uridine Monophosphate (UMP) is Uridine Monophosphate (UMP) is
synthesized firstsynthesized first
•CTP is synthesized from UMPCTP is synthesized from UMP

Pyrimidine ring synthesis completed Pyrimidine ring synthesis completed
first; then attached to ribose-5-first; then attached to ribose-5-
phosphatephosphate
N1, C4, C5, C6 : Aspartate
C2 : HCO3
-
N3 : Glutamine amide Nitrogen

47. 2 ATP + HCO
3
-
+ Glutamine + H
2O
CO
O PO
3
-2
NH
2
Carbamoyl Phosphate
NH
2
C
N
H
CH
CH
2
C
COO
O
HO
O
Carbamoyl Aspartate
HN
C
N
H
CH
CH
2
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
HH
CH
2
OH OH
H H
O
O
2-
O
3P

Orotidine-5'-monophosphate
(OMP)
HN
C
N
CH
CH
C
O
O
HH
CH
2
OH OH
H H
O
O
2-
O
3P

Uridine Monophosphate
(UMP)
2 ADP +
Glutamate +
P
i
Carbamoyl
Phosphate
Synthetase II
Aspartate
Transcarbamoylase
(ATCase)
Aspartate
P
i
H
2O
Dihydroorotase
Quinone
Reduced
Quinone
Dihydroorotate
Dehydrogenase
PRPP PP
i
Orotate Phosphoribosyl
Transferase
CO
2
OMP
Decarboxylase
Pyrimidine Synthesis

48. UMP Synthesis OverviewUMP Synthesis Overview

2 ATPs needed: both used in first step2 ATPs needed: both used in first step
•One transfers phosphate, the other is hydrolyzed to ADP One transfers phosphate, the other is hydrolyzed to ADP
and Piand Pi

2 condensation rxns: form carbamoyl aspartate 2 condensation rxns: form carbamoyl aspartate
and dihydroorotate (intramolecular)and dihydroorotate (intramolecular)

Dihydroorotate dehydrogenase is an Dihydroorotate dehydrogenase is an intra-intra-
mitochondrial mitochondrial enzyme; oxidizing power comes enzyme; oxidizing power comes
from quinone reductionfrom quinone reduction

Attachment of base to ribose ring is catalyzed by Attachment of base to ribose ring is catalyzed by
OPRT; OPRT; PRPP provides ribose-5-PPRPP provides ribose-5-P
•PPPP
ii splits off PRPP – irreversible splits off PRPP – irreversible

Channeling: enzymes 1, 2, and 3 on same chain; Channeling: enzymes 1, 2, and 3 on same chain;
5 and 6 on same chain5 and 6 on same chain

49. OMP DECARBOXYLASE : THE MOST OMP DECARBOXYLASE : THE MOST
CATALYTICALLY PROFICIENT ENZYMECATALYTICALLY PROFICIENT ENZYME

FINAL REACTION OF PYRIMIDINE PATHWAYFINAL REACTION OF PYRIMIDINE PATHWAY

ANOTHER MECHANISM FOR DECARBOXYLATIONANOTHER MECHANISM FOR DECARBOXYLATION

A HIGH ENERGY CARBANION INTERMEDIATE NOT A HIGH ENERGY CARBANION INTERMEDIATE NOT
NEEDEDNEEDED

NO COFACTORS NEEDED !NO COFACTORS NEEDED !

SOME OF THE BINDING ENERGY BETWEEN OMP SOME OF THE BINDING ENERGY BETWEEN OMP
AND THE ACTIVE SITE IS USED TO STABILIZE AND THE ACTIVE SITE IS USED TO STABILIZE
THE TRANSITION STATETHE TRANSITION STATE
•““PREFERENTIAL TRANSITION STATE BINDING”PREFERENTIAL TRANSITION STATE BINDING”

51. UMP UMP  UTP and CTP UTP and CTP

Nucleoside monophosphate kinase Nucleoside monophosphate kinase
catalyzes transfer of Pcatalyzes transfer of Pii to UMP to form UDP; to UMP to form UDP;
nucleoside diphosphate kinase catalyzes nucleoside diphosphate kinase catalyzes
transfer of Ptransfer of Pii from ATP to UDP to form UTP from ATP to UDP to form UTP

CTP formed from UTP via CTP formed from UTP via CTP SynthetaseCTP Synthetase
driven by ATP hydrolysis driven by ATP hydrolysis
•Glutamine provides amide nitrogen for CGlutamine provides amide nitrogen for C4 4
in animalsin animals

53. Regulatory Control of Pyrimidine Regulatory Control of Pyrimidine
SynthesisSynthesis

Differs between bacteria and animalsDiffers between bacteria and animals
•Bacteria – regulation at ATCase rxnBacteria – regulation at ATCase rxn

AnimalsAnimals – regulation at carbamoyl phosphate – regulation at carbamoyl phosphate
synthetase IIsynthetase II
•UDP and UTP inhibit enzyme; ATP and PRPP activate itUDP and UTP inhibit enzyme; ATP and PRPP activate it
•UMP and CMP competitively inhibit OMP DecarboxylaseUMP and CMP competitively inhibit OMP Decarboxylase
*Purine synthesis inhibited by ADP and GDP at *Purine synthesis inhibited by ADP and GDP at
ribose phosphate pyrophosphokinase step, ribose phosphate pyrophosphokinase step,
controlling level of PRPP controlling level of PRPP  also regulates also regulates
pyrimidinespyrimidines

54. Orotic AciduriaOrotic Aciduria

Caused by defect in protein chain with Caused by defect in protein chain with
enzyme activities of last two steps of enzyme activities of last two steps of
pyrimidine synthesispyrimidine synthesis

Increased excretion of orotic acid in Increased excretion of orotic acid in
urine urine

Symptoms: retarded growth; severe Symptoms: retarded growth; severe
anemiaanemia

Only known inherited defect in this Only known inherited defect in this
pathway pathway (all others would be lethal (all others would be lethal
to fetus)to fetus)

Treat with uridine/cytidineTreat with uridine/cytidine

IN-CLASS QUESTION: HOW DOES URIDINE IN-CLASS QUESTION: HOW DOES URIDINE
AND CYTIDINE ADMINISTRATION WORK TO AND CYTIDINE ADMINISTRATION WORK TO
TREAT OROTIC ACIDURIA?TREAT OROTIC ACIDURIA?

55. Degradation of PyrimidinesDegradation of Pyrimidines

CMP and UMP degraded to bases CMP and UMP degraded to bases
similarly to purines similarly to purines
•DephosphorylationDephosphorylation
•DeaminationDeamination
•Glycosidic bond cleavageGlycosidic bond cleavage

Uracil reduced in liver, forming Uracil reduced in liver, forming --
alanine alanine
•Converted to malonyl-CoA Converted to malonyl-CoA  fatty acid fatty acid
synthesis for energy metabolismsynthesis for energy metabolism

56. Deoxyribonucleotide FormationDeoxyribonucleotide Formation

Purine/Pyrimidine degradation are the Purine/Pyrimidine degradation are the
same for ribonucleotides and same for ribonucleotides and
deoxyribonucleotidesdeoxyribonucleotides

Biosynthetic pathways are only for Biosynthetic pathways are only for
ribonucleotide production ribonucleotide production

Deoxyribonucleotides are synthesized from Deoxyribonucleotides are synthesized from
corresponding ribonucleotides corresponding ribonucleotides

57. DNA vs. RNA: REVIEWDNA vs. RNA: REVIEW

DNA composed of deoxyribonucleotidesDNA composed of deoxyribonucleotides

Ribose sugar in DNA lacks hydroxyl group Ribose sugar in DNA lacks hydroxyl group
at 2’ Carbonat 2’ Carbon

Uracil doesn’t (normally) appear in DNAUracil doesn’t (normally) appear in DNA
•Thymine (5-methyluracil) appears insteadThymine (5-methyluracil) appears instead

58. Formation of DeoxyribonucleotidesFormation of Deoxyribonucleotides

Reduction of 2’ carbon done via a Reduction of 2’ carbon done via a free free
radical mechanismradical mechanism catalyzed by catalyzed by
“Ribonucleotide Reductases” “Ribonucleotide Reductases”
•E. coli E. coli RNR reduces ribonucleoside RNR reduces ribonucleoside
diphosphates (NDPs) to deoxyribonucleoside diphosphates (NDPs) to deoxyribonucleoside
diphosphates (dNDPs)diphosphates (dNDPs)

Two subunits: R1 and R2Two subunits: R1 and R2
•A Heterotetramer: (R1)A Heterotetramer: (R1)
22 and (R2) and (R2)
2 2 in vitroin vitro


59. RIBONUCLEOTIDE REDUCTASERIBONUCLEOTIDE REDUCTASE

R1 SUBUNITR1 SUBUNIT
•Three allosteric sitesThree allosteric sites

Specificity SiteSpecificity Site

Hexamerization siteHexamerization site

Activity SiteActivity Site
•Five redox-active –SH groups from cysteinesFive redox-active –SH groups from cysteines

R2 SUBUNITR2 SUBUNIT
•Tyr 122 radicalTyr 122 radical
•Binuclear Fe(III) complexBinuclear Fe(III) complex

60. Ribonucleotide Reductase R2 Ribonucleotide Reductase R2
Subunit Subunit

Fe prosthetic group– binuclear, with each Fe prosthetic group– binuclear, with each
Fe Fe octahedrallyoctahedrally coordinated coordinated
•Fe’s are bridged by OFe’s are bridged by O
-2-2
and carboxyl gp of Glu and carboxyl gp of Glu
115115
•Tyr 122 is close to the Fe(III) complex Tyr 122 is close to the Fe(III) complex 
stabilization of a tyrosyl free-radicalstabilization of a tyrosyl free-radical

During the overall process, a pair of –SH During the overall process, a pair of –SH
groups provides the reducing equivalentsgroups provides the reducing equivalents
•A protein disulfide group is formedA protein disulfide group is formed
•Gets reduced by two other sulfhydryl gps of Gets reduced by two other sulfhydryl gps of
Cys residues in R1Cys residues in R1

61. Chime Exercise
E. coli Ribonucleotide Reductase:
3R1R and 4R1R: R1 subunit
1RIB 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 -helices in one unit of R2
–Tyr 122 residue
–The binuclear Fe (III) complex
–The ligands of the Fe (III) complex
 

62. Mechanism of Ribonucleotide Reductase Mechanism of Ribonucleotide Reductase
ReactionReaction

Free RadicalFree Radical

Involvement of multiple –SH groupsInvolvement of multiple –SH groups

RR is left with a disulfide group that RR is left with a disulfide group that
must be reduced to return to the must be reduced to return to the
original enzymeoriginal enzyme

63. RIBONUCLEOTIDE REDUCTASERIBONUCLEOTIDE REDUCTASE

ACTIVITY IS RESPONSIVE TO LEVEL OF ACTIVITY IS RESPONSIVE TO LEVEL OF
CELLULAR NUCLEOTIDES:CELLULAR NUCLEOTIDES:
•ATP ACTIVATES REDUCTION OFATP ACTIVATES REDUCTION OF

CDPCDP

UDPUDP
•dTTP dTTP

INDUCES GDP REDUCTIONINDUCES GDP REDUCTION

INHIBITS REDUCTION OF CDP. UDPINHIBITS REDUCTION OF CDP. UDP
•dATP INHIBITS REDUCTION OF ALL NUCLEOTIDESdATP INHIBITS REDUCTION OF ALL NUCLEOTIDES
•dGTP dGTP

STIMULATES ADP REDUCTIONSTIMULATES ADP REDUCTION

INHIBITS CDP,UDP,GDP REDUCTIONINHIBITS CDP,UDP,GDP REDUCTION

65. RIBONUCLEOTIDE REDUCTASERIBONUCLEOTIDE REDUCTASE

CATALYTIC ACTIVITY VARIES WITH STATE OF CATALYTIC ACTIVITY VARIES WITH STATE OF
OLIGOMERIZATION:OLIGOMERIZATION:
•WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE WHEN ATP, dATP, dGTP, dTTP BIND TO SPECIFICITY SITE
OF R1 (CATALYTICALLY INACTIVE MONOMER)OF R1 (CATALYTICALLY INACTIVE MONOMER)
 CATALYTICALLY ACTIVE (R1)CATALYTICALLY ACTIVE (R1)
22
•WHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERSWHEN dATP OR ATP BIND TO ACTIVITY SITE OF DIMERS

 TETRAMER FORMATIONTETRAMER FORMATION
(R1)(R1)
4a4a (ACTIVE STATE) == (R1) (ACTIVE STATE) == (R1)
4b4b (INACTIVE) (INACTIVE)
•WHEN ATP BINDS TO HEXAMERIZATION SITEWHEN ATP BINDS TO HEXAMERIZATION SITE
 CATALYTICALLY ACTIVE HEXAMERS (R1)CATALYTICALLY ACTIVE HEXAMERS (R1)
66

66. ThioredoxinThioredoxin

Physiologic reducing agent of RNRPhysiologic reducing agent of RNR

Cys pair can swap H atoms with disulfide Cys pair can swap H atoms with disulfide
formed formed regenerate original enzymeregenerate original enzyme
•Thioredoxin gets oxidized to disulfideThioredoxin gets oxidized to disulfide
Oxidized Thioredoxin gets reduced by NADPH ( final electron acceptor)
mediated by thioredoxin reductase

67. Thymine FormationThymine Formation

Formed by methylating deoxyuridine Formed by methylating deoxyuridine
monophosphate (dUMP) monophosphate (dUMP)

UTP is needed for RNA production, but UTP is needed for RNA production, but
dUTP not needed for DNAdUTP not needed for DNA
•If dUTP produced excessively, would cause If dUTP produced excessively, would cause
substitution errors (dUTP for dTTP)substitution errors (dUTP for dTTP)

dUTP hydrolyzed by dUTPase dUTP hydrolyzed by dUTPase
(dUTP diphosphohydrolase) to dUMP (dUTP diphosphohydrolase) to dUMP 
methylated at C5 to form dTMPmethylated at C5 to form dTMP
rephosphorylate to form dTTPrephosphorylate to form dTTP

68. CHIME EXERCISE: dUTPaseCHIME EXERCISE: dUTPase

1DUD: Deoxyuridine-5'-Nucleotide Hydrolase in a 1DUD: Deoxyuridine-5'-Nucleotide Hydrolase in a
complex with a bound substrate analog, complex with a bound substrate analog,
Deoxyuridine-5'-Diphosphate (dUDP). Deoxyuridine-5'-Diphosphate (dUDP).

Explore dUTPase as follows:Explore dUTPase as follows:
•Find the substrate in its binding siteFind the substrate in its binding site
•Find C5 on the Uracil group. Is there enough room to Find C5 on the Uracil group. Is there enough room to
attach a methyl group to C5?attach a methyl group to C5?
•Locate the ribose 2’ C. What protein group sterically Locate the ribose 2’ C. What protein group sterically
prevents an –OH group from being attached to the 2’ C prevents an –OH group from being attached to the 2’ C
atom?atom?
•Find the H-bond donors and acceptors (to the uracil Find the H-bond donors and acceptors (to the uracil
base) from the protein. What would be the effect on the base) from the protein. What would be the effect on the
H-bonding if the base was changed to cytosine?H-bonding if the base was changed to cytosine?

69. Tetrahydrofolate (THF)Tetrahydrofolate (THF)

Methylation of dUMP catalyzed by Methylation of dUMP catalyzed by
thymidylate synthase thymidylate synthase
•Cofactor: NCofactor: N
55
,N,N
1010
-methylene THF-methylene THF

Oxidized to dihydrofolateOxidized to dihydrofolate

Only known rxn where net oxidation state Only known rxn where net oxidation state
of THF changesof THF changes

THF Regeneration:THF Regeneration:
DHF + NADPH + HDHF + NADPH + H
++
 THF + NADP THF + NADP
++
(enzyme: dihydrofolate (enzyme: dihydrofolate
reductase)reductase)
THF + Serine THF + Serine  N N
55
,N,N
1010
-methylene-THF + Glycine-methylene-THF + Glycine
(enzyme: serine hydroxymethyl transferase)(enzyme: serine hydroxymethyl transferase)

70. dUMP dTMP
NADPH + H
+
NADP
+
SERINE
GLYCINE
REGENERATION OF N
5
,N
10
METHYLENETETRAHYDROFOLATE
DHF
N
5
,N
10
– METHYLENE-THF
THF
dihydrofolate reductase
serine hydroxymethyl
transferase
thymidylate synthase

71. dUMP dTMP
NADPH + H
+
NADP
+
SERINE
GLYCINE
INHIBITORS OF N
5
,N
10
METHYLENETETRAHYDROFOLATE
REGENERATION
DHF
N
5
,N
10
– METHYLENE-THF
THF
dihydrofolate reductase
serine hydroxymethyl
transferase
thymidylate synthase

METHOTREXATE
AMINOPTERIN
TRIMETHOPRIM
FdUMP
X
X

72. Anti-Folate DrugsAnti-Folate Drugs

Cancer cells consume dTMP quickly for Cancer cells consume dTMP quickly for
DNA replicationDNA replication
•Interfere with thymidylate synthase rxn to Interfere with thymidylate synthase rxn to
decrease dTMP production decrease dTMP production

(fluorodeoxyuridylate – irreversible inhibitor) – also (fluorodeoxyuridylate – irreversible inhibitor) – also
affects rapidly growing normal cells (hair follicles, affects rapidly growing normal cells (hair follicles,
bone marrow, immune system, intestinal mucosa)bone marrow, immune system, intestinal mucosa)

Dihydrofolate reductase step can be Dihydrofolate reductase step can be
stopped competitively (DHF analogs)stopped competitively (DHF analogs)
•Anti-Folates: Aminopterin, methotrexate, Anti-Folates: Aminopterin, methotrexate,
trimethoprimtrimethoprim

73. ADENOSINE DEAMINASE DEFICIENCYADENOSINE DEAMINASE DEFICIENCY

IN PURINE DEGRADATION, ADENOSINE IN PURINE DEGRADATION, ADENOSINE 
INOSINEINOSINE
•ENZYME IS ADAENZYME IS ADA

ADA DEFICIENCY RESULTS IN SCIDADA DEFICIENCY RESULTS IN SCID
•““SEVERE COMBINED IMMUNODEFICIENCY”SEVERE COMBINED IMMUNODEFICIENCY”

SELECTIVELY KILLS LYMPHOCYTESSELECTIVELY KILLS LYMPHOCYTES
•BOTH B- AND T-CELLSBOTH B- AND T-CELLS
•MEDIATE MUCH OF IMMUNE RESPONSEMEDIATE MUCH OF IMMUNE RESPONSE

ALL KNOWN ADA MUTANTS STRUCTURALLY ALL KNOWN ADA MUTANTS STRUCTURALLY
PERTURB ACTIVE SITEPERTURB ACTIVE SITE

74. Adenosine DeaminaseAdenosine Deaminase
CHIME Exercise: 2ADACHIME Exercise: 2ADA
Enzyme catalyzing deamination of Adenosine to Inosine Enzyme catalyzing deamination of Adenosine to Inosine
// barrel domain structure barrel domain structure
–““TIM Barrel” – central barrel structure with 8 twisted TIM Barrel” – central barrel structure with 8 twisted
parallel parallel -strands connected by 8 -strands connected by 8 -helical loops-helical loops
–Active site is at bottom of funnel-shaped pocket Active site is at bottom of funnel-shaped pocket
formed by loopsformed by loops
–Found in all glycolytic enzymesFound in all glycolytic enzymes
–Found in proteins that bind and transport metabolitesFound in proteins that bind and transport metabolites

75. ADA DEFICIENCYADA DEFICIENCY
******

IN-CLASS QUESTION: EXPLAIN THE IN-CLASS QUESTION: EXPLAIN THE
BIOCHEMISTRY THAT RESULTS WHEN A PERSON BIOCHEMISTRY THAT RESULTS WHEN A PERSON
HAS ADA DEFICIENCYHAS ADA DEFICIENCY

(HINT: LYMPHOID TISSUE IS VERY ACTIVE IN (HINT: LYMPHOID TISSUE IS VERY ACTIVE IN
DEOXYADENOSINE PHOSPHORYLATION)DEOXYADENOSINE PHOSPHORYLATION)

76. ADA DEFICIENCYADA DEFICIENCY

ONE OF FIRST DISEASES TO BE TREATED WITH ONE OF FIRST DISEASES TO BE TREATED WITH
GENE THERAPYGENE THERAPY

ADA GENE INSERTED INTO LYMPHOCYTES; THEN ADA GENE INSERTED INTO LYMPHOCYTES; THEN
LYMPHOCYTES RETURNED TO PATIENTLYMPHOCYTES RETURNED TO PATIENT

PEG-ADA TREATMENTSPEG-ADA TREATMENTS
•ACTIVITY LASTS 1-2 WEEKSACTIVITY LASTS 1-2 WEEKS