De Novo Synthesis of Purine Nucleotides We use for purine nucleotides the entire glycine molecule atoms 4 57 the amino nitrogen of aspartate atom 1 amide nitrogen of glutamine atoms 3 9 components of the ID: 601104
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Slide1
Nucleic Acid metabolismSlide2
De Novo
Synthesis of
Purine
Nucleotides
We use for
purine
nucleotides the entire
glycine
molecule (atoms 4, 5,7), the amino nitrogen of
aspartate
(atom 1), amide nitrogen of glutamine (atoms 3, 9), components of the
folate
-one-carbon pool(atoms 2, 8), carbon dioxide, ribose 5-P from glucose and a great deal of energy in the form of ATP. In de novo synthesis, IMP is the first nucleotide formed. It is then converted to either AMP or GMP.Slide3
PRPP
Since the
purines
are synthesized as the
ribonucleotides
, (not as the free bases) a necessary prerequisite is the synthesis of the activated form of ribose 5-phosphate. Ribose 5-phosphate reacts with ATP to form
5-Phosphoribosyl-1-pyrophosphate (PRPP)
.Slide4
This reaction occurs in many tissues because PRPP has a number of roles -
purine
and
pyrimidine
nucleotide synthesis, salvage pathways, NAD and NADP formation. The enzyme is heavily controlled by a variety of compounds (
di
- and tri-phosphates, 2,3-DPG), presumably to try to match the synthesis of PRPP to a need for the products in which it ultimately appearsSlide5
Commitment Step
De novo
purine
nucleotide synthesis occurs actively in the
cytosol
of the liver where all of the necessary enzymes are present as a macro-molecular aggregate. The first step is a replacement of the pyrophosphate of PRPP by the amide group of glutamine. The product of this reaction is
5-Phosphoribosylamine
. The amine group that has been placed on carbon 1 of the sugar becomes nitrogen 9 of the ultimate
purine
ring. This is the commitment and rate-limiting step of the pathwaySlide6
Control of
De Novo
Synthesis
Control of
purine
nucleotide synthesis has two phases. Control of the
synthesis as a whole
occurs at the
amidotransferase
step by nucleotide inhibition and/or [PRPP]. The second phase of control is involved with
maintaining an appropriate balance (not equality) between ATP and GTP
. Each one stimulates the synthesis of the other by providing the energy. Feedback inhibition also controls the branched portion as GMP inhibits the conversion of IMP to XMP and AMP inhibits the conversion of IMP to
adenylosuccinate
.Slide7
De Novo
Synthesis of
Pyrimidine
Nucleotides
Since
pyrimidine
molecules are simpler than
purines
, so is their synthesis simpler but is still from readily available components. Glutamine's amide nitrogen and carbon dioxide provide atoms 2 and 3 or the
pyrimidine
ring. They do so, however, after first being converted to
carbamoyl
phosphate. The other four atoms of the ring are supplied by
aspartate
. As is true with
purine
nucleotides, the sugar phosphate portion of the molecule is supplied by PRPP.
Carbamoyl
Phosphate
Pyrimidine
synthesis begins with
carbamoyl
phosphate
synthesized in the
cytosol
of those tissues capable of making
pyrimidines
(highest in spleen, thymus,
GItract
and testes). This uses a different enzyme than the one involved in urea synthesis.
Carbamoyl
phosphate
synthetase
II (CPS II)
prefers glutamine to free ammonia and has no requirement for N-
AcetylglutamateSlide8
Formation of
Orotic
Acid
Carbamoyl
phosphate condenses with
aspartate
in the presence of
aspartate
transcarbamylase
to yield N-
carbamylaspartate
which is then converted to
dihydroorotate
.
In man,
CPSII, asp-
transcarbamylase
, and
dihydroorotase
activities
are part of a
multifunctional protein
.
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
. Note the contrast with
purine
synthesis in which a nucleotide is formed first while
pyrimidines
are first synthesized as the
free base
. Slide9
Salvaging
Purines
As a salvage process though, we are dealing with
purines
. There are two enzymes, A-PRT and HG-PRT.
A-PRT
is not very important because we generate very little adenine. (Remember that the catabolism of adenine nucleotides and nucleosides is through
inosine
).
HG-PRT
, though, is exceptionally important and it is inhibited by both IMP and GMP. This enzyme salvages guanine directly and adenine indirectly. Remember that AMP is generated primarily from IMP, not from free adenineSlide10
Lesch-Nyhan
Syndrome
HG-PRT is deficient in the disease called
Lesch-Nyhan
Syndrome
, a severe neurological disorder whose most blatant clinical manifestation is an uncontrollable self-mutilation.
Lesch-Nyhan
patients have very
high blood uric acid
levels because of an essentially
uncontrolled
de novo
synthesis
. (It can be as much as 20 times the normal rate). There is a significant increase in PRPP levels in various cells and an inability to maintain levels of IMP and GMP via salvage pathways. Both of these factors could lead to an increase in the activity of the
amidotransferase
.Slide11
Purine
Catabolism
The end product of
purine
catabolism in man is
uric acid
. Other mammals have the enzyme
urate
oxidase
and excrete the more soluble
allantoin
as the end product. Man does not have this enzyme so
urate
is the end product for us. Uric acid is formed primarily in the liver and excreted by the kidney into the urine
.Slide12
Bases to Uric Acid
Both adenine and guanine nucleotides converge at the common intermediate
xanthine
. Hypoxanthine, representing the original adenine, is oxidized to
xanthine
by the enzyme
xanthine
oxidase
. Guanine is
deaminated
, with the amino group released as ammonia, to
xanthine
. If this process is occurring in tissues other than liver, most of the ammonia will be transported to the liver as glutamine for ultimate excretion as urea.
Xanthine
, like hypoxanthine, is oxidized by oxygen and
xanthine
oxidase
with the production of hydrogen peroxide. In man, the
urate
is excreted and the hydrogen peroxide is degraded by
catalase
.
Xanthine
oxidase
is present in significant concentration only in liver and intestine. The pathway to the nucleosides, possibly to the free bases, is present in many tissues.Slide13
Gouts and
Hyperuricemia
Both
undissociated
uric acid and the monosodium salt (primary form in blood) are only sparingly soluble. The limited solubility is not ordinarily a problem in urine unless the urine is very acid or has high [Ca
2+
]. [
Urate
salts
coprecipitate
with calcium salts and can form stones in kidney or bladder.] A very high concentration of
urate
in the blood leads to a fairly common group of diseases referred to as gout. The incidence of gout in this country is about 3/1000.
Gout
is a group of pathological conditions associated with markedly elevated levels of
urate
in the blood (3-7 mg/dl normal).
Hyperuricemia
is not always symptomatic, but, in certain individuals, something triggers the deposition of sodium
urate
crystals in joints and tissues. In addition to the extreme pain accompanying acute attacks, repeated attacks lead to destruction of tissues and severe arthritic-like malformations. The term gout should be restricted to
hyperuricemia
with the presence of these
tophaceous
deposits.Slide14
Urate
in the blood could accumulate either through an overproduction and/or an
underexcretion
of uric acid. In gouts caused by an
overproduction
of uric acid, the defects are in the control mechanisms governing the production of - not uric acid itself - but of the nucleotide precursors. The
only major control of
urate
production that we know so far is the availability of substrates (nucleotides, nucleosides or free bases)
.
One approach to the treatment of gout is the drug
allopurinol
, an isomer of hypoxanthine.
Allopurinol
is a substrate for
xanthine
oxidase
, but the product binds so tightly that the enzyme is now unable to oxidized its normal substrate. Uric acid production is diminished and
xanthine
and hypoxanthine levels in the blood rise. These are more soluble than
urate
and are less likely to deposit as crystals in the joints. Another approach is to stimulate the secretion of
urate
in the urine.Slide15
Pyrimidine
Catabolism
In contrast to
purines
,
pyrimidines
undergo ring cleavage and the usual end products of catabolism are beta-amino acids plus ammonia and carbon dioxide.
Pyrimidines
from nucleic acids or the energy pool are acted upon by
nucleotidases
and
pyrimidine
nucleoside
phosphorylase
to yield the free bases. The 4-amino group of both cytosine and 5-methyl cytosine is released as ammonia.
Ring Cleavage
In order for the rings to be cleaved, they must first be
reduced by NADPH
. Atoms 2 and 3 of both rings are released as ammonia and carbon dioxide. The rest of the ring is left as a
beta-amino acid
. Beta-amino
isobutyrate
from thymine or 5-methyl cytosine is largely excreted. Beta-
alanine
from cytosine or
uracil
may either be excreted or incorporated into the brain and muscle
dipeptides
,
carnosine
(his-beta-ala) or
anserine
(methyl his-beta-ala).Slide16Slide17