BIOCHEMISTRY DR.
TAMPUS
TOPIC OUTLINE
LECTURE TITLE (CAMBRIA 11, BOLD, ALL CAPS)
I. Nucleotides
II. Metabolism of Purines and Pyrimidines
III. Nucleic Acid Strcuture and Function
IV. DNA Organization, Replication, Repair
CHAPTER 32: NUCLEOTIDES
BIOMEDICAL IMPORTANCE
Purine and pyrimidine nucleotides
 precursors of nucleic acids
 participate in metabolic function (energy metabolism, protein
synthesis, regulation of enzyme activity and signal transduction)
 Form portion of many enzymes when linked to vitamin or vitamin
derivative
 Principal donor and acceptor of phosphoryl group in metabolism
 ATP, ADP are principal players in energy transduction that
accompanies metabolic interconversion and oxidative
phosphorylation
 When linked to sugars or lipids  NUCLEOSIDES constitute
key biosynthetic intermediates.
 sugar derivative UDP – glucose, UDP- galactose
participate in sugar interconversion and
biosynthesis of starch and glycogen
 Nucleoside-lipid derivative CDP-acylglycerol in
lipid biosynthesis
Roles that nucleotides perform in metabolic regulation:
- ATP dependent phosphorylation of key metabolic enzyme
- Allosteric regulate of enzyme by ATP, ADP, AMP, CTP
- Control of ADP of rate of oxidative phosphorylation
- cAMP, cGMP serve as second messenger in hormonally
regulated events
- GTP, GDP key role in cascade of events that characterized
signal transduction pathway
Medical Applications:
 use of synthetic purine and pyrimidine analog that contain
halogens, thiols/ additional N₂ atoms in chemotherapy of cancer/AID
 suppressors of immune response during organ transplantation
CHEMISTRY OF PURINES, PYRIMIDINES, NUCLEOSIDES, &
NUCLEOTIDES
Purines & Pyrimidines Are Heterocyclic Compounds
- Nitrogen containing heterocycles that contain C, N₂
- Pyrimidine is smaller molecule compared to purine molecule
- 6-atom rings are numbered in opposite direction
- Purines or pyrimidines with(-NH₂) are weak bases; pKa value 3-4
- Proton associated with ring N₂ typically N₁ of adenine, N₇ of
guanine, N₃ of cytosin
- Planar character facilitate their close association or stacking =
stabilizes double-stranded DNA
- Oxo and amino group exhibit keto-enol and amino-imine
tautomerism
Nucleosides Are N-Glycosides
 NUCLEOTIDES are derivatives of purines and pyrimidines that
have a sugar linked to a ring nitrogen of a purine or pyrimidine
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 Numerals with a prime (eg, 2′ or 3′) distinguish atoms of the sugar
from those of the heterocycle.
 The sugar in ribonucleosides is D-ribose & in
deoxyribonucleosides is 2-deoxy-D-ribose.
 Both sugars are linked to the heterocycle by a B-N-
glycosidic bond, almost always to the N-1 of a pyrimidine
or to the N-9 of a purine
Nucleotides Are Phosphorylated Nucleosides
Mononucleotide – nucleoside with phosphoryl group esterified to a
hydroxyl group of sugar.
 the 3’ and 5’ nucleotide are nucleosides with phosphoryl group
on 3’- / 5’-hydroxyl group of sugar, respectively (most nucleotides
are 5’ so it is omitted when naming)
UMP and dAMP: represent nucleotideswith a phosphoryl group on
C-5 of the pentose.
 Additional phosphoryl group, ligated by acid anhydride
bonds to phosporyl group of mononucleotide, form
nucleoside diphosphate and triphosphate
*SKIPPED 2 SUBHEADERS
* Heterocylic N-Glycosides Exist as Syn and Anti Conformers &
Modification of Polynucleotides Can Generate Additional
Structures
Nucleotides Are Polyfunctional Acids
Primary and secondary phosphoryl group of nucleoside bear
significant negative charge at physiologic pH
primary – pKa – 1
secondary – pKa – 6.2 ( can serve as proton donor /
proton acceptor
* therefore bear significant negative charge at physiologic pH.
2ndary phosphoryl group pka values: can serve as proton
donor/acceptors at pH values approx.. 2 or more units above/below
neutrality
Nucleotides Absorb Ultraviolet Light
The conjugated double bonds of purine and pyrimidine
derivatives absorb ultraviolet light.
 spectra is pH dependent
 at pH 7.0 all the common nucleotides absorb light at a wv close to
260 nm.
 mutagenic effect of UV light is due to absorption by nucleotides
in DNA that result in chemical modification
Nucleotides Serve Diverse Physiologic Functions
- Precursor of nucleic acid, ATP, GTP, UTP, CTP and
derivatives
- Role of ATP as principal biologic transducer of free energy
cAMP second messenger
1 mmol/L: mean intracellular conc. of ATP (most abundant free
nucleotide in mammalian cell)
Little camp is required: 1 nmol/L
Other examples:
Adenosine 3′-phosphate-5′-phosphosulfate: sulphate donor for
sulphated proteoglycans & for sufate conjugates of drugs
SAM: methyl group donor
GTP: allosteric regulator and as anenergy source for protein
synthesis
CGMP: second messenger in response to nitric oxide during
relaxation of somooth muscle
 CHAPTER 32-35

UDP-sugar derivatives: participate in sugar epimerizations  Catalyzed by phosphodiesterase; hydrolysis of

and in biosynthesis of glycogen,glucosyl disaccharides, and the
oligosaccharides of glycoproteins and proteoglycans.
UDP glucoronic acid: forms urinary glycorunide conjugates of
bilirubin and many drugs including aspirin
CTP participate in biosynthesis of phosphoglyceride, sphingomyelin
and other substitute sphingosides
*MANY COENZYMES incorporate nucleotides as well as structures
similar to purine and pyrimidine metabolism.
Nucleoside Triphosphates Have High Group Transfer Potential
- Nucleotide triphosphate have 2 acid anhydride bonds and
one-ester bond thus has high group transfer potential ( -7
Kcal/mol) thus can function as group transfer reagent
(most common: γ-phosphoryl group) and transfer of a
nucleotide monophosphate moiety with release of PP₁
- Cleavage of an acid anhydride bond coupled with highly
endergonic process of covalent bond synthesis the
polymerization of nucleoside triphosphate form nucleic
acid
phosphodiester bond of DNA occurs over long period of
time
RNA – less stable than DNA
 2’hydroxyl group of RNA (absent in DNA) function as nucleophile
during hydrolysis of the 3’,5’- phosphodiester bond
Posttranslational modification of preformed polynucleotides can
generate structures like:
Pseudouridine – nucleoside in which D-ribose is linked to C-5 of
uracil by C to C bond
Psuedouridylic acid – rearrangement of a UMP of a preformed tRNA
Thymine monophosphate – methylation of S-adenosyl-methionine
of UMP of preformed tRNA
* Polynucleotides Are Directional Macromolecules

SYNTHETHIC NUCLEOTIDE ANALOGS

ARE USED IN CHEMOTHERAPY
Application in clinical medicine of synthetic analogs of purine,
pyrimidine, nucleoside and nucleotides modified in heterocyclic ring
or in the sugar moiety:
 toxic effects reflect inhibition of enzyme inhibition of
enzyme essential for nucleic acid synthesis incorporation
into nucleic acid with resulting disruption of base-pairing
 oncologists employ: 5-fluro-or 5-iodouracil, 3-
deoxyuridine, 6-thioguarine and 6-mercaptopurine ,etc =
incorporated into DNA prior to cell division
 allopurinol (analog) – treatment for hyperuricemia and
gout; inhibit purine synthesis and xanthine oxidase activity
 Anticancer drugs:
Cytarabine – chemotherapy for cancer
Azathioprine – catabolized to 6-mercaptopurine;
employed during organ transplant to suppress
immunologic rejection

SYNTHETHIC, Non-Hydrolyzable Nucleoside Triphosphate

Analogs Serve as Research Tools
 Used to distinguish effect of nucleotides due to phosphoryl
transfer from effect mediated by occupancy of allosteric nucleotide-
binding sited on regulated enzyme

DNA & RNA are POLYNUCLEOTIDES

 5’-phosphoryl group of mononucleatide esterify second –
hydroxyl group forming phosphodiester
 3’-OH of pentose of second nucleotide forming
dinucleotide
 Pentose moieties are linked by a 3’,5’-phosphodiester
bond to form the backbone of RNA and DNA
 The formation of dinucleotide maybe represented as
elimination of water between 2 mononucleotides
 Hydrolysis of phosphodiester bond is thermodynamically
favored

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CHAPTER 33: Metabolism of Purine &
Pyrimidine Nucleotides
BIOMEDICAL IMPORTANCE
 Dietary purine and pyrimidine are not incorporated
directly into tissue nucleic acid
 Nucleic acid, ATP, NAD⁺, COENZYME A are synthesized
from amphibolic intermediates
 Injected purine/pyrimidine analogs, including anticancer
drugs may be incorporated into DNA
 Biosynthesis of purine and pyrimidine ribonucleotides
triphosphate are precisely regulated
 Coordinated feedback mechanism ensure appropriate
quantities depend on physiologic needs ( growth / tissue
regeneration)
 Unlike the low solubility of uric acid formed by catabolism
of purine, the end products of pyrimidine catabolism (CO2,
ammonia, B-alanine) are highly WATER SOLUBLE
DISEASES:
A. Due to abnormal purine metabolism:
GOUT, LESCH-NYHAN, ADENOSINE DEAMINASE DEF, PURINE
NUCLEOSIDE PHOSPHORYLASE DEF.
B. Diseases that involve ABN in pyrimidine metabolism:
RARE, e.g OROTIC ACIDURIAS, β-hydroxybutyric aciduria due to
deficient in dihydropyrimidine dehydrogenase, thymimeria with
associated β-alanine and β-aminoisobutyrate impairment
 * B-hydroxybutyric aciduria: genetic disorder of pyrimidine
catabolism; d/t total/partial def of dihydropyrimidine
dehydrogenase. AKA: combined uraciluria-thyminuria
PURINES AND PYRIMIDINES ARE DIETARY NONESSENTIAL
Normal human tissues: can synthesize both from amphibolic
intermedates  degradeded in GIT  mononucleotides
absorbed/converted to purine/pyrimidine bases.
 PURINE bases are oxidized to uric acid  abs/exc in urine
*little/non of both is incorp to tissue nucleic acids, injected
compounds are incorp, the incorp of injected [3H] thymidine into
newly synthesized DNA thus can be used to measure rate of DNA
SYNTHESIS
BIOSYNTHESIS OF PURINE NUCLEOTIDES
 All forms of life synthesize purine and pyrimidine
nucleotides except parasitic protozoa
 To achieve homeostasis: intracellular mechanisms sense
and regulate the pool sizes of NTPs which rise during
growth/ regeneration
 3 processes of purine nucleotide biosynthesis in
decreasing importance:
1. Synthesis from amphibolic intermediates
2. Phosphoribosylation of purines.
3. Phosphorylation of purine nucleosides.
INOSINE MONOPHOSPHATE (IMP) IS SYNTHESIZED
FROM AMPHIBOLIC INTERMEDIATES
5-phosphoribosyl 5-pyrophosphate (PRPP)
 First intermediated in de novo pathway of purine
biosynthesis
 Also the intermediate in synthesis of pyrimidine
nucleotide, NAD⁺, NADP⁺
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CHAPTER 32-35
► leads to formation of inosine monophosphate (IMP) through
phosphoryl transfer from ATP convert AMP and GMP to ADP and
GDP.
*ADP by oxidative phosphorylaton converted to ATP; while GDP
involves a second phosphoryl transfer from ATP form GTP
NOT IN PPT:
*Multifunctional Catalysts Participate in Purine Nucleotide
Biosynthesis
*Antifolate Drugs & Glutamine Analogs Block Purine Nucleotide
Biosynthesis
“SALVAGE REACTIONS” CONVERT PURINES &
THEIR NUCLEOSIDES TO MONONUCLEOTIDES
Salvage reaction
 Conversion of purine, their nucleotisides and
deoxyribonucleosides to mononucleotide
 Requires less energy than de novo synthesis
The more impt mechanism involves: Phosphorylation by PRPP of a
free purine to form purine 5’mononucleotide (Pu-RP).
Phosphoryl transfer from PRPP converts:
→ Adenine catalyzed by adenosine-phosphoribosyl transferase to
adenomonophosphate
→ Hypoxanthine catalyze by hypoxanthine phosphoribosyl
transferase to inosine monophosphate
→Guanine catalyze by phosphoribisyl transferase to guanine
monophosphate
Second salvage mechanism
Involves phosphate transfer from ATP - purine ribonucleoside (Pu-R)
 Phosphorylation of purine nucleotides catalyzed by adenosine
kinase convert adenosine and deoxyadenosin to AMP and dAMP
 Deoxycytidine kinase phosphorylates deoxycytidine and
2’deoxyguanosine to form dCMP and dGMP
Liver: major site of purine nucleotide biosynthesis, provides purines
and purine nucleosides for salvage and for utilization by tissues
incapable of biosynthesis.
Human brain tissue: low level of PRPP glutamyl amidotransferase
and depends in part on exogenous purines
RBCs and PMNs: cannot synthesize 5-phosphoribosylamine and also
utilize exogenous purines
HEPATIC PURINE BIOSYNTHESIS: STRINGENTLY REGULATED
AMP & GMP Feedback Regulate PRPP Glutamyl Amidotransferase
IMP biosynthesis: energetically expensive; consume ATP, glycerine,
glutamine, aspartate and reduced tetrahydrofolate derivatives
Regulation:
 PRPP concentration – overall determinant of de novo
purine nucleotide biosynthesis. Rate of synthesis is
dependent on ribose5-phosphate and activity of PRPP
synthase whose activity is feedback inhibited by AMP,
ADP, GMP, GDP
 AMP feedback inhibits adenylosuccinate synthase
 GMP inhibits IMP dehydrogenase
Cross regulation between pathway of IMP metabolism serve to
balance biosynthesis of purine nucleotide then there is deficiency of
the other nucleotide

- conversion of IMP to adenylsuccinate enroute to AMP require GTP
- conversion of xanthinylate to GMP require ATP
 AMP and GMP inhibits hypoxanthine-guanin
phosphoribosyl transferase
 GMP feedback inhibits PRPP glutanyl amidotransferase
Not in PPT:
*REDUCTION OF RIBONUCLEOSIDE DIPHOSPHATES FORMS
DEOXYRIBONUCLEOSIDE DIPHOSPHATES
BIOSYNTHESIS OF PYRIMIDINE NUCLEOTIDES
Pyrimidine biosynthesis:
Carbamoyl phosphatate synthase II – catalyst for initial reaction;
cytosolic
*carbamoyl phosphate synthase ii: mitochon, for urea synthesis
Compartmentation provides an independent pool of carbamoyl
phosphate for each process unlike in purine biosynthesis where
PRPP serves as a scaffold for assembly of the purine ring.
PRPP: participates in pyrimidine biosynthesis only subsequent to
assembly of the pyrimidine ring.
Multifunctional Proteins Catalyze the Early
Reactions of Pyrimidine Biosynthesis
First 5 out of 6 enzymes have/reside on multifunctional
polypeptides- close proximity of active sites on the multifunctional
polypeptide facilitates efficient channeling of intermediates for
pyrimidine biosynthesis
THE DEOXYRIBONUCLEOSIDES OF
URACIL & CYTOSINE ARE SALVAGED
Salvage pathway:
 Adenine, guanine, hypoxanthine are released during
turnover of nucleic acid (mRNA) are reconverted to
nucleotide triphosphate
 Convert pyrimidine ribonucleotide uridine and cytidine
and pyrimidine deoxyribonucleoside thymidine and
deoxycytidine to their respective nucleotide
* Phosphoryltransferases (kinases) catalyze transfer…
* Methotrexate Blocks Reduction of Dihydrofolate
 the rxn catalysed by thymidylate synthase is the only reaction of
pyrimidine nucleotide biosynthesis that requires a tetrahydrofolate
derivative.
*Allopurinol and the anticancer drug 5-fluorouracil: alternate
substances for orotate phosphoribosyltransferase.
REGULATION OF PYRIMIDINE NUCLEOTIDE BIOSYNTHESIS
 Allosteric regulation – first and second enzyme activities
 Carbamoyl phosphate synthase II – inhibited by UTP and
purine nucleotide and activated by PRPP
 Aspartate transcarmoylase is inhibited by CTP; activated
by ATP
 First 3 and last 2 enzymes: regulated by coordinate
repression and derepression
Purine & Pyrimidine Nucleotide Biosynthesis Are
Coordinately Regulated
PRPP synthase – forms the precursors essential to both processes;
feedback inhibited by purine and pyrimidine nucleotide
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HUMANS CATABOLIZE PURINES TO URIC ACID
Humans convert adenosine and guanosine  uric acid
Adenosine  inosine; enzyme: adenosine deaminase
Uric acid  water soluble allantoin; enz: uricase
Humans lack uricase so EP is uric acid
DISORDERS OF PURINE CATABOLISM
GOUT: genetic defects in PRPP synthase
 Gouty arthritis  when serum urate levels exceed solubility
limit, sodim urate crystalizes in soft tissues and joints 
inflamm rxn
Purine deficiency: rare
Hyperuricemia: differentiated based on whether patients excrete
Normal or Excess quantities of total urates.
 Lesch-Nyhan syndrome – overproduction; hyperurecemia
characterized by uric acid lithiasis, bizarre syndrome of self
mutilation;
DEFECT: hypoxanthine-guanine phosphoribosyl transferase ;
INC in intracell PRP  purine overproduction
Mutations that dec or abolish hypoxanthine-guanine
phosphoribosyltransferase activity include: deletion, frame-
shift mutation, aberrant mRNA splicing, base subs
 Von Gierke Disease: purine overproduction and
hyperuricemia; G6PPD deficiency; occurs secondary to
enhanced generation of PRPP precursor ribose 5-phosphate;
an assoc. lactic acidosis elevates the renal threshold for urate
 INC total body urates
 Adenosine deaminase deficiency – immunodeficiency disease
T-cell and B-cells are sparse and dysfunctional
 Purine nucleoside phosphorylase deficiency – severe defect
of T cell but B-cell is normal immune dysfunction result from
accommulation of dGTP and dATP that inhibits ribonucleotide
reductase thus deplete cells of DNA precursor
Catabolism of pyrimidine result to
highly water soluble products
 CO₂, NH₃, β-alanine, β-aminoisobutyrate
leukemia and severe x-ray radiation exposure leads to increase
excretion of β-aminoisobutyrate due to increase destruction of DNA
*READ TABLE PAGE 356
Enzyme defects in pyrimidine catabolism:
 Β-hydroxybutyric aciduria – total or partial deficiency of
dihyropyrimidine dehyrogenase
 Β-amino acid metabolism – combine uraciluria and
thyminuria; impaired formation of β-alanine and β-
aminoisobutyrate
 Orotic aciduria with Reyes syndrome – severely damage
mitochondria
type I – deficiency of orotate phosphocarbosyl transferase
and orotidylate decarboxylase
type II – defect in orotidylate decarboxylase only
Deficiency in liver mitochondrial ornithine transcarbomoylase –
increase excretion of orotic acid, uracil, uridine; excess carbamoyl
phosphate exits to cytosol where it stimulates pyrimidine nucleotide
biosynthesis
Allopurinol – alternative substrate for orotate phospharibosyl
transferase, competes with orotic acid
6-azauridine - competitively inhibits orotidylate decarboxylase
leading to increase excretion of orotic acid and urotidine
 CHAPTER 32-35

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