BIOCHEMISTRY DR.

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

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

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

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 & ► leads to formation of inosine monophosphate (IMP) through
Pyrimidine Nucleotides phosphoryl transfer from ATP convert AMP and GMP to ADP and
GDP.
*ADP by oxidative phosphorylaton converted to ATP; while GDP
BIOMEDICAL IMPORTANCE
involves a second phosphoryl transfer from ATP form GTP
 Dietary purine and pyrimidine are not incorporated
directly into tissue nucleic acid
NOT IN PPT:
 Nucleic acid, ATP, NAD⁺, COENZYME A are synthesized
*Multifunctional Catalysts Participate in Purine Nucleotide
from amphibolic intermediates
Biosynthesis
 Injected purine/pyrimidine analogs, including anticancer
*Antifolate Drugs & Glutamine Analogs Block Purine Nucleotide
drugs may be incorporated into DNA
Biosynthesis
 Biosynthesis of purine and pyrimidine ribonucleotides
triphosphate are precisely regulated
 Coordinated feedback mechanism ensure appropriate “SALVAGE REACTIONS” CONVERT PURINES &
quantities depend on physiologic needs ( growth / tissue THEIR NUCLEOSIDES TO MONONUCLEOTIDES
regeneration) Salvage reaction
 Unlike the low solubility of uric acid formed by catabolism  Conversion of purine, their nucleotisides and
of purine, the end products of pyrimidine catabolism (CO2, deoxyribonucleosides to mononucleotide
ammonia, B-alanine) are highly WATER SOLUBLE  Requires less energy than de novo synthesis
DISEASES: The more impt mechanism involves: Phosphorylation by PRPP of a
A. Due to abnormal purine metabolism: free purine to form purine 5’mononucleotide (Pu-RP).
GOUT, LESCH-NYHAN, ADENOSINE DEAMINASE DEF, PURINE Phosphoryl transfer from PRPP converts:
NUCLEOSIDE PHOSPHORYLASE DEF. → Adenine catalyzed by adenosine-phosphoribosyl transferase to
B. Diseases that involve ABN in pyrimidine metabolism: adenomonophosphate
RARE, e.g OROTIC ACIDURIAS, β-hydroxybutyric aciduria due to → Hypoxanthine catalyze by hypoxanthine phosphoribosyl
deficient in dihydropyrimidine dehydrogenase, thymimeria with transferase to inosine monophosphate
associated β-alanine and β-aminoisobutyrate impairment →Guanine catalyze by phosphoribisyl transferase to guanine
 * B-hydroxybutyric aciduria: genetic disorder of pyrimidine monophosphate
catabolism; d/t total/partial def of dihydropyrimidine
dehydrogenase. AKA: combined uraciluria-thyminuria Second salvage mechanism
Involves phosphate transfer from ATP - purine ribonucleoside (Pu-R)
PURINES AND PYRIMIDINES ARE DIETARY NONESSENTIAL  Phosphorylation of purine nucleotides catalyzed by adenosine
Normal human tissues: can synthesize both from amphibolic kinase convert adenosine and deoxyadenosin to AMP and dAMP
intermedates  degradeded in GIT  mononucleotides  Deoxycytidine kinase phosphorylates deoxycytidine and
absorbed/converted to purine/pyrimidine bases. 2’deoxyguanosine to form dCMP and dGMP
 PURINE bases are oxidized to uric acid  abs/exc in urine
*little/non of both is incorp to tissue nucleic acids, injected Liver: major site of purine nucleotide biosynthesis, provides purines
compounds are incorp, the incorp of injected [3H] thymidine into and purine nucleosides for salvage and for utilization by tissues
newly synthesized DNA thus can be used to measure rate of DNA incapable of biosynthesis.
SYNTHESIS Human brain tissue: low level of PRPP glutamyl amidotransferase
and depends in part on exogenous purines
BIOSYNTHESIS OF PURINE NUCLEOTIDES RBCs and PMNs: cannot synthesize 5-phosphoribosylamine and also
utilize exogenous purines
 All forms of life synthesize purine and pyrimidine
nucleotides except parasitic protozoa
 To achieve homeostasis: intracellular mechanisms sense HEPATIC PURINE BIOSYNTHESIS: STRINGENTLY REGULATED
and regulate the pool sizes of NTPs which rise during AMP & GMP Feedback Regulate PRPP Glutamyl Amidotransferase
growth/ regeneration
 3 processes of purine nucleotide biosynthesis in IMP biosynthesis: energetically expensive; consume ATP, glycerine,
decreasing importance: glutamine, aspartate and reduced tetrahydrofolate derivatives
1. Synthesis from amphibolic intermediates Regulation:
2. Phosphoribosylation of purines.  PRPP concentration – overall determinant of de novo
3. Phosphorylation of purine nucleosides. purine nucleotide biosynthesis. Rate of synthesis is
dependent on ribose5-phosphate and activity of PRPP
synthase whose activity is feedback inhibited by AMP,
INOSINE MONOPHOSPHATE (IMP) IS SYNTHESIZED
ADP, GMP, GDP
FROM AMPHIBOLIC INTERMEDIATES
 AMP feedback inhibits adenylosuccinate synthase
5-phosphoribosyl 5-pyrophosphate (PRPP)
 GMP inhibits IMP dehydrogenase
 First intermediated in de novo pathway of purine
Cross regulation between pathway of IMP metabolism serve to
biosynthesis
balance biosynthesis of purine nucleotide then there is deficiency of
 Also the intermediate in synthesis of pyrimidine
the other nucleotide
nucleotide, NAD⁺, NADP⁺

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- conversion of IMP to adenylsuccinate enroute to AMP require GTP HUMANS CATABOLIZE PURINES TO URIC ACID
- conversion of xanthinylate to GMP require ATP Humans convert adenosine and guanosine  uric acid
 AMP and GMP inhibits hypoxanthine-guanin Adenosine  inosine; enzyme: adenosine deaminase
phosphoribosyl transferase Uric acid  water soluble allantoin; enz: uricase
 GMP feedback inhibits PRPP glutanyl amidotransferase Humans lack uricase so EP is uric acid

Not in PPT: DISORDERS OF PURINE CATABOLISM

*REDUCTION OF RIBONUCLEOSIDE DIPHOSPHATES FORMS GOUT: genetic defects in PRPP synthase
DEOXYRIBONUCLEOSIDE DIPHOSPHATES  Gouty arthritis  when serum urate levels exceed solubility
limit, sodim urate crystalizes in soft tissues and joints 
BIOSYNTHESIS OF PYRIMIDINE NUCLEOTIDES inflamm rxn
Pyrimidine biosynthesis: Purine deficiency: rare
Carbamoyl phosphatate synthase II – catalyst for initial reaction; Hyperuricemia: differentiated based on whether patients excrete
cytosolic Normal or Excess quantities of total urates.
*carbamoyl phosphate synthase ii: mitochon, for urea synthesis  Lesch-Nyhan syndrome – overproduction; hyperurecemia
Compartmentation provides an independent pool of carbamoyl characterized by uric acid lithiasis, bizarre syndrome of self
phosphate for each process unlike in purine biosynthesis where mutilation;
PRPP serves as a scaffold for assembly of the purine ring. DEFECT: hypoxanthine-guanine phosphoribosyl transferase ;
INC in intracell PRP  purine overproduction
PRPP: participates in pyrimidine biosynthesis only subsequent to
Mutations that dec or abolish hypoxanthine-guanine
assembly of the pyrimidine ring.
phosphoribosyltransferase activity include: deletion, frame-
shift mutation, aberrant mRNA splicing, base subs
Multifunctional Proteins Catalyze the Early
 Von Gierke Disease: purine overproduction and
Reactions of Pyrimidine Biosynthesis hyperuricemia; G6PPD deficiency; occurs secondary to
First 5 out of 6 enzymes have/reside on multifunctional enhanced generation of PRPP precursor ribose 5-phosphate;
polypeptides- close proximity of active sites on the multifunctional an assoc. lactic acidosis elevates the renal threshold for urate
polypeptide facilitates efficient channeling of intermediates for  INC total body urates
pyrimidine biosynthesis  Adenosine deaminase deficiency – immunodeficiency disease
T-cell and B-cells are sparse and dysfunctional
THE DEOXYRIBONUCLEOSIDES OF  Purine nucleoside phosphorylase deficiency – severe defect
URACIL & CYTOSINE ARE SALVAGED of T cell but B-cell is normal immune dysfunction result from
Salvage pathway: accommulation of dGTP and dATP that inhibits ribonucleotide
 Adenine, guanine, hypoxanthine are released during reductase thus deplete cells of DNA precursor
turnover of nucleic acid (mRNA) are reconverted to Catabolism of pyrimidine result to
nucleotide triphosphate highly water soluble products
 Convert pyrimidine ribonucleotide uridine and cytidine  CO₂, NH₃, β-alanine, β-aminoisobutyrate
and pyrimidine deoxyribonucleoside thymidine and leukemia and severe x-ray radiation exposure leads to increase
deoxycytidine to their respective nucleotide excretion of β-aminoisobutyrate due to increase destruction of DNA
* Phosphoryltransferases (kinases) catalyze transfer… *READ TABLE PAGE 356
* Methotrexate Blocks Reduction of Dihydrofolate
Enzyme defects in pyrimidine catabolism:
 the rxn catalysed by thymidylate synthase is the only reaction of
 Β-hydroxybutyric aciduria – total or partial deficiency of
pyrimidine nucleotide biosynthesis that requires a tetrahydrofolate
dihyropyrimidine dehyrogenase
derivative.
 Β-amino acid metabolism – combine uraciluria and
*Allopurinol and the anticancer drug 5-fluorouracil: alternate thyminuria; impaired formation of β-alanine and β-
substances for orotate phosphoribosyltransferase. aminoisobutyrate
 Orotic aciduria with Reyes syndrome – severely damage
REGULATION OF PYRIMIDINE NUCLEOTIDE BIOSYNTHESIS mitochondria
 Allosteric regulation – first and second enzyme activities type I – deficiency of orotate phosphocarbosyl transferase
 Carbamoyl phosphate synthase II – inhibited by UTP and and orotidylate decarboxylase
purine nucleotide and activated by PRPP type II – defect in orotidylate decarboxylase only
 Aspartate transcarmoylase is inhibited by CTP; activated
by ATP Deficiency in liver mitochondrial ornithine transcarbomoylase –
 First 3 and last 2 enzymes: regulated by coordinate increase excretion of orotic acid, uracil, uridine; excess carbamoyl
repression and derepression phosphate exits to cytosol where it stimulates pyrimidine nucleotide
biosynthesis
Purine & Pyrimidine Nucleotide Biosynthesis Are
Allopurinol – alternative substrate for orotate phospharibosyl
Coordinately Regulated
transferase, competes with orotic acid
PRPP synthase – forms the precursors essential to both processes;
feedback inhibited by purine and pyrimidine nucleotide
6-azauridine - competitively inhibits orotidylate decarboxylase
leading to increase excretion of orotic acid and urotidine

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