1. Nucleotides: Synthesis and Degradation

2. Nitrogenous Bases Planar, aromatic, and heterocyclic
Derived from purine or pyrimidine
Numbering of bases is “unprimed”

3. Nucleic Acid Bases
Pyrimidines
Purines

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

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

6. 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

7. Nucleosides

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

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

10. Nucleotides RNA (ribonucleic acid) is a polymer of ribonucleotides
DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides
Both deoxy- and ribonucleotides contain Adenine, Guanine and Cytosine
Ribonucleotides contain Uracil
Deoxyribonucleotides contain Thymine

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

12. Naming Conventions Nucleosides: Nucleotides:
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

13. 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

14. 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

15. Purine Nucleotides
Get broken down into Uric Acid (a purine) Buchanan (mid 1900s) showed where purine ring components came from:
N1: Aspartate Amine
C2, C8: Formate
N3, N9: Glutamine
C4, C5, N7: Glycine
C6: Bicarbonate Ion

16. Purine Nucleotide Synthesis

17. 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 a to b (anomers specifying OH positioning on C1 with respect to C4 group)
In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)

18. Coupling of Reactions
Hydrolyzing a phosphate from ATP is relatively easy
G°’= 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”)

19. 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

20. 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

21. IMP Conversion to AMP

22. IMP Conversion to GMP

23. 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
Rate of AMP production increases with increasing concentrations of GTP; rate of GMP production increases with increasing concentrations of ATP

24. 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

25. 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 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.

26. 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)

27. 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

28. Adenosine Degradation

29. 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

30. 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

31. 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

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

33. 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

34. Purine Salvage Adenine + PRPP  AMP + PPi
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 !

35. 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.

36. 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.

37. 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

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

42. ALCOHOL CONSUMPTION AND GOUT
Choi HK, Atkinson K, Karlson EW et al “Alcohol intake and risk of incident gout in men:
a prospective study”. Lancet 363:

43. 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

44. 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

45. 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?

46. 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

47. Pyrimidine Synthesis

48. 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

49. 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”

51. 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 in animals

53. 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

54. 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?

55. Degradation of Pyrimidines
CMP and UMP degraded to bases similarly to purines
Dephosphorylation
Deamination
Glycosidic bond cleavage
Uracil reduced in liver, forming b-alanine
Converted to malonyl-CoA  fatty acid synthesis for energy metabolism

56. 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

57. 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

58. 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

59. 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

60. 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

61. E. coli Ribonucleotide Reductase:
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 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

63. 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

65. 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

66. 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

67. 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

68. 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?

69. 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)

70. REGENERATION OF N5,N10 METHYLENETETRAHYDROFOLATE
dUMP
dTMP
thymidylate synthase
DHF
N5,N10 – METHYLENE-THF
NADPH + H+
GLYCINE
dihydrofolate reductase
serine hydroxymethyl
transferase
NADP+
SERINE
THF

71. INHIBITORS OF N5,N10 METHYLENETETRAHYDROFOLATE REGENERATION
dUMP
dTMP
thymidylate synthase
DHF
N5,N10 – METHYLENE-THF X
NADPH + H+
GLYCINE
FdUMP
dihydrofolate reductase
serine hydroxymethyl
transferase
NADP+
SERINE X
THF
METHOTREXATE
AMINOPTERIN
TRIMETHOPRIM

72. 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

73. 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

74. Adenosine Deaminase CHIME Exercise: 2ADA
Enzyme catalyzing deamination of Adenosine to Inosine
a/b barrel domain structure
“TIM Barrel” – central barrel structure with 8 twisted parallel b-strands connected by a-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

75. 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)

76. 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