NUCLEOPROTEINS

METABOLISM
 Nucleotides are organic molecules that serve as the monomers
of nucleic acids like DNA and RNA. The building blocks of nucleotides
are composed of a nitrogenous base, a five-carbon sugar (ribose
or deoxyribose and phosphate group.
Nucleotides are needed for DNA and RNA synthesis (DNA replication and
transcription) and for energy transfer. Nucleoside triphosphates (ATP and GTP)
provide energy for reactions that would otherwise be extremely unfavorable in the
cell. Ribose 5-phosphate for nucleotide synthesis is derived from the hexose
monophosphate shunt and is activated by the addition of pyrophosphate from ATP,
forming phosphoribosyl pyrophosphate (PRPP) using PRPP synthetase. Cells
synthesize nucleotides in 2 ways: de novo synthesis and salvage pathways. • In de
novo synthesis, which occurs predominantly in the liver, purines and pyrimidines are
synthesized from smaller precursors, and PRPP is added to the pathway at some
point. • In the salvage pathways, preformed purine and pyrimidine bases can be
converted into nucleotides by salvage enzymes distinct from those of de novo
synthesis. Purine and pyrimidine bases for salvage enzymes may arise from: –
Synthesis in the liver and transport to other tissues – Digestion of endogenous
nucleic acids (cell death, RNA turnover

In many cells, the capacity for de novo synthesis to supply purines and pyrimidines is
insufficient, and the salvage pathway is essential for adequate nucleotide
synthesis. In Lesch-Nyhan disease, an enzyme for purine salvage (hypoxanthine
guanine phosphoribosyl pyrophosphate transferase, HPRT) is absent or deficient.
People with this genetic deficiency have CNS deterioration, mental retardation, and
spastic cerebral palsy associated with compulsive self-mutilation. Cells in the basal
ganglia of the brain (fine motor control) normally have very high HPRT activity.
Patients also all have hyperuricemia because purines cannot be salvaged, causing
gout.
 There are two kinds of nitrogen-containing bases - purines and pyrimidines.
Purines consist of a six-membered and a five-membered nitrogen-containing
ring, fused together. Pyridmidines have only a sixmembered nitrogen-containing
ring. There are 2 purines and 3 pyrimidines nitrogen-containing bases.
Three nucleobases found in nucleic acids - cytosine (C), thymine (T),
and uracil(U), are pyrimidine derivatives:
 In DNA, the purines adenine (A)
and guanine (G) pair up with the
pyrimidines thymine (T) and cytosine (C),
respectively.
adenine:thymine and guanine:cytosine

In RNA, the complement of adenine (A)

is uracil (U) instead of thymine (T), so the
pairs that form are
adenine:uracil and guanine:cytosine.
Biomedical importance:
1. The heterocyclic bases purines and pyrimidines are the
parent molecules of nucleotides, which are present in
every cell.
2. DNA is present in the nucleus forming chromosomes
which are responsible for transmission of genetic
characters including regulation of protein synthesis.
RNA is present in both nucleus and cytoplasm and has
a central role process of protein synthesis.
3. Structure of viruses is almost nucleoproteins.
4. Synthetic analogs of normally occurring nucleotides may
find application the treatment of cancer. These analogs
can act as enzyme inhibitors or replace the natural
nucleotides in nucleic acids synthesis in this way viral
growth and cancer cell replication are inhibited.
 Digestion of nucleoproteins:

- The protein is successively removed

from the nucleoproteins by pepsin and
trypsin.
- The nucleic acidsare split by a series of
enzymes of the pancreatic juice (the
nucleases: DNA-ase and RNA-ase).
Nucleases (DNA-аse і RNA-
ase) decompose nucleic acids
into oligonucletides.
Oligonucleotide
Phosphodiesterases decompose oligonucleotides to
mononucleotides

Nucleotides
structure
Phosphatases
– enzymes
that split off
phosphoric
acid and
formed -
nucleosides
Nucleosidases –
enzymes, that
catalyse decomposition
nucleosides to nitrogenous
bases and pentoses
Nitrogenous bases
DECOMPOSITION OF MONONUCLEOTIDE

NH 2
Phosphatases N
N
OH N N
O P O CH2 O Nucleosidases
OH
H H
H H

OH OH

Adenosine mononucleotide
DECOMPOSITION OF NUCLEIC ACIDS IN
INTESTINE AND TISSUE

Nucleoproteins (nucleic
acids + proteins)

Pepsin, gastricsin, HCl

Nucleic acids + Histones, protamines

Nucleases (DNA-ases, RNA-ases)

Oligonucleotides
Phosphodiesterases
Mononucleotides
Phosphatases

Nuclesides + Phosphoric acid

Nucleosidases
Nitrogenous
+ Pentose
bases
 DESTINY OF NITROGENOUS BASES, PENTOSES AND
PHOSPHORIC ACIDS IN THE ORGANISM

Nitrogenous oxidation to the end products

bases

oxidation with energy formation;

synthesis of nucleotided;
Pentoses
synthesis of hexoses;
synthesis of coenzymes

phosphorilation;
ATP synthesis;
Phosphoric acid synthesis of phospholipids;
buffer systems;
constituent of bones, cartilages
Absorption:
• Nucleotides and nucleosides are fairly
absorbed from the small intestine. They
are partly incorporated into tissue nucleic
acids and partly catabolized in the liver.
• Purines and pyrimidines are poorly
absorbed from the small intestine. They
are mostly catabolized in the liver, only
adenine may partly incorporated into
tissue nucleic acids.
 Synthesis of purine bases
Origin of atoms in purine molecule
CO2

H Glycine
C N
Aspartate N 6
5
C 7 CH Methenil-H4-
1 8
folate
2 4
Formil-H4- HC 3 C 9
folate
N N
H
R

Glutamine
 The biosynthetic origins of purine
ring atoms
N1 - arises from the amino group
of Aspartate
C2 and C8 - originate from folate
(vitamin B10)
N3 and N9 - are contributed by the amide
group of Glutamine
C4, C5 and N7 - are derived from Glycine
C6 - comes from HCO3− (CO2)
•
Figure I-18-1. Nucleotide Synthesis by
Salvage and De Novo Pathway
PURINE SYNTHESIS Purines are synthesized de novo beginning with PRPP. The
most important enzyme is PRPP amidotransferase, which catalyzes the first and rate-
limiting reaction of the pathway. It is inhibited by the 3 purine nucleotide end products
AMP, GMP, and IMP.

The drugs allopurinol (used for gout) and 6-mercaptopurine (antineoplastic) also inhibit
PRPP amidotransferase. These drugs are purine analogs which must be converted to
their respective nucleotides by HGPRT within cells. • The amino acids glycine,
aspartate, and glutamine are used in purine synthesis. • Tetrahydrofolate is required
for synthesis of all the purines. • Inosine monophosphate (contains the purine base
hypoxanthine) is the precursor for AMP and GMP.

Bridge to Microbiology Protozoan and multicellular parasites and many obligate

parasites, such as Chlamydia, cannot synthesize purines de novo because they lack
the necessary genes in the purine pathway. However, they have elaborate salvage
mechanisms for acquiring purines from the host to synthesize their own nucleic acids
to grow.
PURINE CATABOLISM AND THE SALVAGE ENZYME HGPRT Excess
purine nucleotides or those released from DNA and RNA by nucleases are
catabolized first to nucleosides (loss of Pi) and then to free purine bases
(release of ribose or deoxyribose). Excess nucleoside monophosphates
may accumulate when:
• RNA is normally digested by nucleases (mRNAs and other types of
RNAs are continuously turned over in normal cells).
• Dying cells release DNA and RNA, which is digested by nucleases.
• The concentration of free Pi decreases as it may in galactosemia,
hereditary fructose intolerance, and glucose-6-phosphatase deficiency.
Salvage enzymes recycle normally about 90% of these purines, and 10%
are converted to uric acid and excreted in urine.
When purine catabolism is increased significantly, a person is at risk for
developing hyperuricemia and potentially gout. Purine catabolism to uric
acid and salvage of the purine bases hypoxanthine (derived from
adenosine) and guanine are shown below.
 Synthesis of Purine
Nucleotides
Ribosphosphate is formed in pentose-
phosphate pathway from glucose
Purine ring is synthesized on ribose-5-
phosphate by the way of gradual
adding of nitrogen and carbon atoms
and cyclization.
The way of biosynthesis consist of 11
reactions.
 Synthesis of Purine
Nucleotides
Two ways of biosynthesis:
-de novo – formation of purine
nucleotides from simple acyclic
precursors (in liver)
-salvage (reserve) pathway – using
of purine bases formed in the
decomposition of nucleotides (in the
out-of-liver tissues)
de novo – formation of purine nucleotides
Catabo
lism of
purine
nucleo
tides
Catabolism of purine
nucleotides
Catabolism
of purine
nucleotides
Uric acid
formation
Adenosine Deaminase Deficiency. Adenosine deaminase (ADA) deficiency, an
autosomal recessive disorder, causes a type of severe combined immunodeficiency
(SCID). Lacking both B-cell and T-cell function, children are multiply infected with
many organisms (Pneumocystis carinii, Candida) and do not survive without
treatment. Enzyme replacement therapy and bone marrow transplantation may be
used. Experimental gene therapy trials have not yet yielded completely successful
cures. High levels of dATP accumulate in red cells of ADA patients and inhibit
ribonucleotide reductase, thereby inhibiting the production of other essential
deoxynucleotide precursors for DNA synthesis (see Figure I-18-3). Although it is
believed that the impaired DNA synthesis contributes to dysfunction of T cells and B
cells, it is not known why the main effects are limited to these cell types.

Bridge to Pharmacology Thiazide diuretics (hydrochlorothiazide and

chlorthalidone) may cause hyperuricemia.

Bridge to Pathology Treatment of large tumors with chemotherapeutic regimens

or radiation may cause “turnor lysis syndrome” and excessive excretion of uric
acid, resulting in gout. The cause of the excessive uric acid is the destruction of
the cancer cell’s nucleic acid into purines undergoing turnover
0.5-1 g of uric acid is formed daily in the
organism and exreated with urine.
Normal concentration of uric acid in blood:
in man – 0.24-0.50 mmol/L
in women – 0,16 – 0,40 mmol/l
Uric acid – poorly soluble in water
High levels of uric acid is called hyperuricemia and can
lead to gout.
Hyperuricemia:
- inherited (primary),
- gained (secondary).
- alimentary
Hyperuricemia and Gout Hyperuricemia may be produced by overproduction
of uric acid or underexcretion of uric acid by the kidneys. Hyperuricemia may
progress to acute and chronic gouty arthritis if uric acid (monosodium urate) is
deposited in joints and surrounding soft tissue, where it causes inflammation.
Uric acid is produced from excess endogenous purines as shown in Figure I-
18-5, and is also produced from dietary purines (digestion of nucleic acid in the
intestine) by intestinal epithelia. Both sources of uric acid are transported in the
blood to the kidneys for excretion in urine. Allopurinol inhibits xanthine oxidase
and also can reduce purine synthesis by inhibiting PRPP amidotransferase,
provided HGPRT is active . Hyperuricemia and gout often accompany the
following conditions:
• Lesch-Nyhan syndrome (no purine salvage)
• Partial deficiency of HGPRT
• Alcoholism (lactate and urate compete for same transport system in the
kidney)
• Glucose 6-phosphatase deficiency
• Hereditary fructose intolerance (aldolase B deficiency)
• Galactose 1-phosphate uridyl transferase deficiency (galactosemia)
• Mutations in PRPP synthetase that lower Km In the last 2 diseases,
phosphorylated sugars accumulate, decreasing the available Pi and increasing
AMP (which cannot be phosphorylated to ADP and ATP). The excess AMP is
converted to uric acid.
Clinical Correlate Gout Acute gouty arthritis, seen most commonly in males, results
from precipitation of monosodium urate crystals in joints. The crystals, identified as
negatively birefringent and needle-shaped, initiate neutrophil-mediated and acute
inflammation, often first affecting the big toe. Chronic gout may manifest over time as
tophi (deposits of monosodium urate) in soft tissue around joints, leading to chronic
inflammation involving granulomas.
• Acute attacks of gout are treated with colchicine or indomethacin to reduce the
inflammation.
• Chronic hyperuricemia, because of underexcretion, is treated with a uricosuric drug
(probenecid).
• Overproduction of uric acid and chronic gout are treated with allo purinol.
Gout and Lesch-Nyhan syndrome – inherited
diseases accompanied with primary
hyperuricemia and crystallization of uric acid
and salts in joints, cartilages and kidneys.
Salts of uric acid – urates.
Symptoms:
-joints inflammation, acute pain
-renal stones

-tophuses.
Uric acid crystal deposits in the joint cause
inflammation of the joint leading to pain,
redness, heat, and swelling.
• Secondary hyperuricemia: in radiation
injury, blood diseases, tumors,
toxemia, kidney diseases.
• Аlimentary (hyperconsumption of
meat, strong coffee, tea)
 URIC ACID - is the end product of purine
metabolism.
- Hyperuricaemia is associated with a tendency to
form crystals of monosodium urate causing
- Clinical gout (due to the deposition of
monosodium urate crystals in the cartilage,
synovium and synovial fluid of joints),
- Renal calculi
- Tophi (accretions of sodium urate in soft tissues)
- Acute urate nephropathy (due to sudden
increases in urate production leading to
widespread crystallisation in the renal tubules).
Gout:
accumula-
tion salts
of uric
acid in
joints
Gout: accumulation of
uric acid salts in joints
Gout: tophuses
– accumulation
of uric acid
salts in soft
tissue, under
skin.
Gout: kidney stones.
Gout
 Ivan Hobachevsky – Doctor of
Medicine, Professor, Head of Medical
Chemistry Department, Dean of
Faculty of Medicine at Charles
University in Prague, explored the
causes and pathogenesis of gout,
mechanisms of catabolism of
mononucleotides, which are
constituents of nucleic acids. First
synthesized uric acid, creatin,
discovered enzyme xanthine oxidase,
developed methods for determining
proteins and purine bases.

Ivan Horbachevsky
uric acid
Lesch-Nyhan Syndrom (LNS): is a inherited disorder
caused by a deficiency of the enzyme hypoxanthine-
guanine phosphoribosyltransferase.
LNS is present at birth in baby boys.
Hypoxanthine and guanine are not used in the salvage
pathway of purine nucleotides synthesis.
Hypoxanthine and guanine are not utilizied repeatedly
but converted into uric acid.
Symptoms:
-severe gout;
-severe mental and physical
problems;
-self-mutilating behaviors.
Lesch-Nyhan syndrom:
• gout-like swelling in some joints
• kidney and bladder stones
• delayed motor development
• bizarre
• sinuous movements
• increased deep tendon reflexes
• self-destructive behavior (chewing off fingertips and lips)
Introduction to Lesch-Nyhan Syndrome
Treatment: allopurinol – competitive
inhibitor of xanthine oxidase
Allopurinol, developed over 30 years ago
by Elion, weakly inhibits xanthine
oxidase. It is an analog of hypoxanthine
that is hydroxylated by xanthine
oxireductase at the 2-position to give
oxipurinol.
Oxipurinol has been supposed to bind
tightly to the reduced molybdenum ion in
the enzyme and, thus, inhibits uric acid
synthesis
SYNTHESIS OF PYRIMIDINE NUCLEOTIDES

Origin of atoms in pyrimidine molecule

Enzyme names:

1. Carbamoyl phosphate synthetase II

2. aspartate transcarbamylase
3. dihydroorotate dehydrogenase
4. orotate phosphoribosyltransferase
5. orotidine-5'-phosphate carboxylase
 Pyrimidine synthesis begins with carbamoyl
phosphate synthesized in the cytosol of those
tissues capable of making pyrimidines (highest
in spleen, thymus, GItract and testes).
Carbamoyl phosphate synthetase II (CPS II)
prefers glutamine to free ammonia
Carbamoyl phosphate condenses with aspartate in
the presence of aspartate transcarbamylase to
yield N-carbamylaspartate which is then
converted to dihydroorotate.

•
Oxidation of the ring by a complex, poorly understood
enzyme produces the free pyrimidine, orotic acid. This
enzyme is located on the outer face of the inner
mitochondrial membrane, in contrast to the other enzymes
which are cytosolic.
Orotic acid is converted to its nucleotide with
phosphoribosyltransferase. OMP is
then converted sequentially - not in a
branched pathway - to the other pyrimidine
nucleotides.
Decarboxylation of OMP gives UMP. O-PRT and
OMP decarboxylase are also a multifunctional
protein. In reaction amination UMP convert to
CMP
• The primary end product of pyrimidine synthesis is UMP. In the conversion of UMP to
dTMP, 3 important enzymes are ribonucleotide reductase, thymidylate synthase, and

dihydrofolate reductase; all are targets of antineoplastic drugs .

• Table I-18-1. Important Enzymes of Pyrimidine Synthesis

Enzyme Function Drug

Ribonucleotide reductase Reduces all NDPs to Hydroxyurea (S phase)

dNDPs for DNA reductase
synthesis

Thymidylate synthase Methylates dUMP to 5-Fluorouracil (S phase)

dTMP Requires THF

Dihydrofolate reductase Converts DHF to THF Methotrexate (eukaryotic)

(DHFR) Without DHFR, (S phase) Trimethoprim
thymidylate synthesis will (prokaryotic)
eventually stop Pyrimethamine
(protozoal)
• Orotic aciduria is an autosomal recessive disorder
caused by a defect in uridine monophosphate (UMP)
synthase. This enzyme contains two activities, orotate
phosphoribosyltransferase and orotidine
decarboxylase. The lack of pyrimidines impairs nucleic
acid synthesis needed for hematopoiesis, explaining the
megaloblastic anemia in this infant. Orotic acid
accumulates and spills into the urine, resulting in orotic
acid crystals and orotic acid urinary obstruction. The
presence of orotic acid in urine might suggest that the
defect could be ornithine transcarbamylase (OTC)
deficiency, but the lack of hyperammonemia rules out a
defect in the urea cycle. Uridine administration relieves the
symptoms by bypassing the defect in the pyrimidine
pathway. Uridine is salvaged to UMP, which feedback-
inhibits carbamoyl phosphate synthase-2, preventing orotic
acid formation.
• Ribonucleotide Reductase
• Ribonucleotide reductase is required for
the formation of the deoxyribonucleotides
for DNA synthesis. • All 4 nucleotide
substrates must be diphosphates. • dADP
and dATP strongly inhibit ribonucleotide
reductase. • Hydroxyurea, an anticancer
drug, blocks DNA synthesis indirectly by
inhibiting ribonucleotide r

• Figure I-18-3. Ribonucleotide Reductaseeductase

 NH2 NH2

N N

Dis-
O N O N
H H
Цитозин
cytosine
1 Тимін
thimine

integ-
/2O2
NH3
NADPH + H+
O
dehydrogenase

ration
HN
NADP+
O N
H O

of
Урацил
uracile CH3
NADPH + H+ HN H
dehydrogenase H
NADP+ O N H
O COO– H

pyri-
H Дигідротимін
dihydrothimine
hydrolase NH2 CH2
HN H H2O
H C CH2 hydrolase
O N N

midine
H H2O O
H H
COO–
Дигідроурацил
dihydrouracile -Уреїдопропіонат CH3
N-carbomoil -alanine
(N-карбамоїл--аланін) NH2 C
H

nucle-
C CH2
H2O N
O H
-Аланін
-alanine H2O
+ – -Уреїдоізобутират
H3N CH2 CH2 COO N-carbomoil -
(N-карбамоїл-
hydrolase

otides
-аміноізобутират)
aminoisobutirate
O2

CH3COO– CO2 + NH3 H3N+ CH2 CH COO–

CH3
-Аміноізобутират
-aminoisobutirate
Catabolism of the pyrimidine nucleotides

CMP and UMP are degraded to β-alanine and

NH3 and CO2 . TMP degraded to β-
aminoisobutyrate.
The β-alanine and β-aminoisobutyrate serve
as -NH2 donors in transamination of α-
ketoglutarate to glutamate.
NH3 and CO2 used in biosynthesis of urea.
β-alanine – structural component of CoA.
β-aminoisobutyrate convert into succinil CoA.
 OROTACIDURIA

inherited disorder of pyrimidine

synthesis caused by a deficiency of the
enzyme of orotate-phosphoribosyl-
transferase and decarboxylase.
Symptoms:
–excess of orotic acid and its excretion
with urine (1.0-1.5 g)
-mental and physical retardation
-megaloblastic anemia
TREATMENT OF
OROTACIDURIA

Taking of
uridin
during the
whole life