BIOSYNTHESIS AND CATABOLISM OF PURINE NUCLEOTIDES. DETERMINATION OF THE END
PRODUCTS OF THEIR METABOLISM. BIOSYNTHESIS AND CATABOLISM OF PYRIMIDINE
NUCLEOTIDES. DISORDERS OF NUCLEOTIDES METABOLISM
One of the important specialized pathways of a number
of amino acids is the synthesis of purine and pyrimidine nucleotides. These
nucleotides are important for a number of reasons. Most of them, not just ATP,
are the sources of energy that drive most of our reactions. ATP is the most
commonly used source but GTP is used in protein synthesis as well as a few
other reactions. UTP is the source of energy for activating glucose and
galactose. CTP is an energy source in lipid metabolism. AMP is part of the
structure of some of the coenzymes like NAD and Coenzyme A. And, of course, the
nucleotides are part of nucleic acids. Neither the bases nor the nucleotides
are required dietary components. We can both synthesize them de novo and
salvage and reuse those we already have.
Nitrogen
Bases
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 six-membered nitrogen-containing ring. There are 4 purines and 4
pyrimidines that are of concern to us.
Purines
http://www.youtube.com/watch?v=uOAsqECXVco
Adenine and guanine are found in both DNA and RNA. Hypoxanthine and
xanthine are not incorporated into the nucleic acids as they are being
synthesized but are important intermediates in the synthesis and degradation of
the purine nucleotides.
http://www.youtube.com/watch?v=PHOjrY3zYdM
Pyrimidines
http://www.youtube.com/watch?v=KXr8bM69Nq4
Cytosine is found in both DNA and RNA. Uracil is found only in RNA.
Thymine is normally found in DNA. Sometimes tRNA will contain some thymine as
well as uracil.
http://www.youtube.com/watch?v=S8DMoDJ8FWA
Nucleosides
If a sugar, either ribose or 2-deoxyribose,
is added to a nitrogen base, the resulting compound is called a nucleoside.
Carbon 1 of the sugar is attached to nitrogen 9 of a purine base or to nitrogen
1 of a pyrimidine base. The names of purine nucleosides end in -osine
and the names of pyrimidine nucleosides end in -idine. The
convention is to number the ring atoms of the base normally and to use l', etc.
to distinguish the ring atoms of the sugar. Unless otherwise specificed, the
sugar is assumed to be ribose. To indicate that the sugar is 2'-deoxyribose, a d-
is placed before the name.
·
Adenosine
·
Guanosine
·
Inosine - the base in inosine is hypoxanthine
·
Uridine
·
Thymidine
·
Cytidine
Nucleotides
Adding one or more phosphates to the sugar portion of a nucleoside
results in a nucleotide. Generally, the phosphate is in ester linkage to
carbon 5' of the sugar. If more than one phosphate is present, they are
generally in acid anhydride linkages to each other. If such is the case, no
position designation in the name is required. If the phosphate is in any other
position, however, the position must be designated. For example, 3'-5' cAMP
indicates that a phosphate is in ester linkage to both the 3' and 5' hydroxyl
groups of an adenosine molecule and forms a cyclic structure. 2'-GMP would
indicate that a phosphate is in ester linkage to the 2' hydroxyl group of a
guanosine. Some
representative names are:
·
AMP
= adenosine monophosphate = adenylic acid
·
CDP
= cytidine diphosphate
·
dGTP
= deoxy guanosine triphosphate
·
dTTP = deoxy thymidine triphosphate
(more commonly designated TTP)
·
cAMP = 3'-5' cyclic adenosine
monophosphate
http://www.youtube.com/watch?v=hW9EmUN-wsc&feature=related
NOMENCLATURE
OF NUCLEIC BASES, NUCLEOSIDES, AND NUCLEOTIDES
|
Nucleobase |
Nucleoside |
Nucleotide 5’-monophosphate |
|
Adenine Guanine Thymine Cytosine Uracil Hypoxanthine Xanthine |
Adenosine Guanosine Thymidine Cytidine Uridine Inosine Xanthosine |
Adenosine 5’-monophosphate (adenylate, AMP) Guanosine 5’-monophosphate (guanylate, GMP) Thymidine 5’-monophosphate (thymidylate,
TMP) Cytidine 5’-monophosphate (cytidylate, CMP) Uridine 5’-monophosphate (uridylate, UMP) Inosine 5’-monophosphate (inosinate, IMP) Xanthosine 5’-monophosphate (xanthylate ,
XMP) |
Polynucleotides
Nucleotides are joined together by 3'-5'
phosphodiester bonds to form polynucleotides. Polymerization of ribonucleotides
will produce an RNA while polymerization of deoxyribonucleotides leads to DNA.
Most, but not all, nucleic acids in the cell are associated with
protein. Dietary nucleoprotein is degraded by pancreatic enzymes and tissue nucleoprotein
by lysosomal enzymes. After dissociation of the protein and nucleic acid, the
protein is metabolized like any other protein.
The nucleic acids are hydrolyzed randomly by nucleases
to yield a mixture of polynucleotides. These are further cleaved by phosphodiesterases
(exonucleases) to a mixture of the mononucleotides. The specificity of the
pancreatic nucleotidases gives the 3'-nucleotides and that of the lysosomal
nucleotidases gives the biologically important 5'-nucleotides.
The nucleotides are hydrolyzed by nucleotidases
to give the nucleosides and Pi. This is probably the end product in
the intestine with the nucleosides being the primary form absorbed. In at least
some tissues, the nucleosides undergo phosphorolysis with nucleoside
phosphorylases to yield the base and ribose 1-P (or deoxyribose 1-P). Since
R 1-P and R 5-P are in equilibrium, the sugar phosphate can either be
reincorporated into nucleotides or metabolized via the Hexose Monophosphate
Pathway. The purine and pyrimidine bases released are either degraded or
salvaged for reincorporation into nucleotides.
There is significant turnover of all kinds of RNA
as well as the nucleotide pool. DNA doesn't turnover but portions of the
molecule are excised as part of a repair process.
Purine and pyrimidines from tissue turnover which
are not salvaged are catabolized and excreted. Little dietary purine is used
and that which is absorbed is largely catabolized as well. Catabolism of purines
and pyrimidines occurs in a less useful fashion than did the catabolism of
amino acids in that we do not derive any significant amount of energy from the
catabolism of purines and pyrimidines. Pyrimidine catabolism, however, does
produce beta-alanine, and the endproduct of purine catabolism, which is uric
acid in man, may serve as a scavenger of reactive oxygen species.
The metabolic requirements for the nucleotides and their cognate bases
can be met by both dietary intake or synthesis de novo from low
molecular weight precursors. Indeed, the ability to salvage nucleotides from
sources within the body alleviates any nutritional requirement for nucleotides,
thus the purine and pyrimidine bases are not required in the diet. The salvage
pathways are a major source of nucleotides for synthesis of DNA, RNA and enzyme
co-factors.
Extracellular hydrolysis of ingested nucleic acids occurs through the
concerted actions of endonucleases, phosphodiesterases and nucleoside
phosphorylases. Endonucleases degrade DNA and RNA at internal sites leading to
the production of oligonucleotides. Oligonucleotides are further digested by
phosphodiesterases that act from the ends inward yielding free nucleosides. The
bases are hydrolyzed from nucleosides by the action of phosphorylases that
yield ribose-1-phosphate and free bases. If the nucleosides and/or bases are
not re-utilized the purine bases are further degraded to uric acid and the
pyrimidines to β-aminoiosobutyrate,
NH3 and CO2.
Purine and pyrimidine bases which are not
degraded are recycled - i.e. reincorporated into nucleotides. This
recycling, however, is not sufficient to meet total body requirements and so
some de novo synthesis is essential. There are definite tissue
differences in the ability to carry out de novo synthesis. De novo
synthesis of purines is most active in liver. Non-hepatic tissues generally
have limited or even no de novo synthesis. Pyrimidine synthesis occurs
in a variety of tissues. For purines, especially, non-hepatic tissues rely
heavily on preformed bases - those salvaged from their own intracellular
turnover supplemented by bases synthesized in the liver and delivered to
tissues via the blood.
"Salvage" of purines is reasonable in
most cells because xanthine oxidase, the key enzyme in taking the purines all
of the way to uric acid, is significantly active only in liver and intestine.
The bases generated by turnover in non-hepatic tissues are not readily degraded
to uric acid in those tissues and, therefore, are available for salvage. The liver
probably does less salvage but is very active in de novo synthesis - not
so much for itself but to help supply the peripheral tissues.
De novo synthesis of
both purine and pyrimidine nucleotides occurs from readily available
components.
Phosphoribosyl pyrophosphate (PRPP) is important in both, and in these
pathways the structure of ribose is retained in the product nucleotide, in
contrast to its fate in the tryptophan and histidine biosynthetic pathways
discussed earlier. An amino acid is an important precursor in each type of
pathway: glycine for purines and aspartate for pyrimidines. Glutamine again is
the most important source of amino groups — in five different steps in the de
novo pathways. Aspartate is also used as the source of an amino group in the
purine pathways, in two steps. Two other features deserve mention. First, there
is evidence, especially in the de novo purine pathway, that the enzymes are
present as large, multienzyme complexes in the cell, a recurring theme in our
discussion of metabolism. Second, the cellular pools of nucleotides (other than
ATP) are quite small, perhaps 1% or less of the amounts required to synthesize
the cell’s DNA.
Therefore, cells must continue to synthesize nucleotides during nucleic
acid synthesis, and in some cases nucleotide synthesis may limit the rates of
DNA replication and transcription. Because of the importance of these processes
in dividing cells, agents that inhibit nucleotide synthesis have become
particularly important to modern medicine. We examine here the biosynthetic
pathways of purine and pyrimidine nucleotides and their regulation, the
formation of the deoxynucleotides, and the degradation of purines and
pyrimidines to uric acid and urea. We end with a discussion of chemotherapeutic
agentsthat affect nucleotide synthesis.
De
Novo
Synthesis of Purine Nucleotides
The two
parent purine nucleotides of nucleic acids are adenosine-monophosphate (AMP;
adenylate) and guanosine-monophosphate (GMP; guanylate), containing the purine
bases adenine and guanine. Figure shows the origin of the carbon and nitrogen
atoms of the purine ring system, as determined by John Buchanan using isotopic
tracer experiments in birds. The detailed pathway of purine biosynthesis was
worked out primarily by Buchanan and G. Robert Greenberg in the 1950s.
Synthesis of the first fully formed purine
nucleotide, inosine monophosphate, IMP begins with 5-phospho-α-ribosyl-1-pyrophosphate,
PRPP. Through a series of reactions utilizing ATP, tetrahydrofolate (THF)
derivatives, glutamine, glycine and aspartate this pathway yields IMP. The rate
limiting reaction is catalyzed by glutamine PRPP amidotransferase, enzyme
indicated by
Enzyme
names:
1. glutamine
phosphoribosylpyrophosphate amidotransferase
2. glycinamide
ribotide synthase
3. glycinamide
ribotide transformylase
4.
formylglycinamide synthase
5.
aminoimidazole ribotide synthase
6.
aminoimidazole ribotide carboxylase
7.
succinylaminoimidazolecarboxamide ribotide synthase
8.
adenylosuccinate lyase
9.
aminoimidazole carboxamide ribotide transformylase
10.
IMP cyclohydrolase
In the first committed step of the pathway, an
amino group donated by glutamine is attached at C-1 of PRPP.
The resulting
5-phosphoribosylamine is highly unstable, with a half-life of 30 seconds
at pH 7.5. The purine ring is subsequently built up on this structure. The
pathway described here is identical in all organisms, with the exception of one
step that differs in higher eukaryotes as noted below.
The second
step is the addition of three atoms from glycine (step 2 ). An ATP is consumed
to activate the glycine carboxyl group (in the form of an acyl phosphate) for
this condensation reaction:
The added
glycine amino group is then formylated by N10- formyltetrahydrofolate
(step 3 ):
A nitrogen is
contributed by glutamine (step 4 ):
Before dehydration
and ring closure yield the five-membered imidazole ring of the purine nucleus,
as 5-aminoimidazole ribonucleotide (AIR; step 5).
At this
point, three of the six atoms needed for the second ring in the purine
structure are in place. To complete the process, a carboxyl group is first
added (step 6 ). This carboxylation is unusual in that it does not require
biotin, but instead uses the bicarbonate generally present in aqueous
solutions. A rearrangement transfers the carboxylate from the exocyclic amino
group to position 4 of the imidazole ring (step 7 ).
Steps 6 and 7
are found only in bacteria and fungi. In higher eukaryotes, including humans,
the 5-aminoimidazole ribonucleotide product of step 5 is carboxylated directly
to carboxyaminoimidazole ribonucleotide in one step instead of two (step 6a).
The enzyme catalyzing this reaction is AIR carboxylase.
Aspartate now
donates its amino group in two steps ( 8 and 9 ): formation of an amide bond,
followed by elimination of the carbon skeleton of aspartate (as fumarate).
Recall that aspartate plays an analogous role
in two steps of the urea cycle. The final carbon is contributed by N10-formyltetrahydrofolate
(step 10 ),
and a second ring
closure takes place to yield the second fused ring of the purine nucleus (step
11).
The first
intermediate with a complete purine ring is inosinate (IMP).
Synthesis of AMP and GMP from
IMP
As in the
tryptophan and histidine biosynthetic pathways, the enzymes of IMP synthesis
appear to be organized as large, multienzyme complexes in the cell. Once again,
evidence comes from the existence of single polypeptides with several
functions, some catalyzing nonsequential steps in the pathway. In eukaryotic
cells ranging from yeast to fruit flies to chickens, steps 1 , 3 , and 5 are
catalyzed by a multifunctional protein. An additional multifunctional protein
catalyzes steps 10 and
In bacteria,
these activities are found on separate proteins, but a large noncovalent
complex may exist in these cells. The channeling of reaction intermediates from
one enzyme to the next permitted by these complexes is probably especially
important for unstable intermediates such as 5-phosphoribosylamine.
Conversion of
inosinate to adenylate requires the insertion of an amino group derived from
aspartate; this takes place in two reactions similar to those used to introduce
N-1 of the purine ring, (steps 8 and 9 ). A crucial difference is that GTP
rather than ATP is the source of the high-energy phosphate in synthesizing
adenylosuccinate.
Guanylate is formed
by the NAD1-requiring oxidation of inosinate at C-2, followed by addition of an
amino group derived from glutamine. ATP is cleaved to AMP and PPi in the final
step.
REGULATION OF
PURINE NUCLEOTIDE BIOSYNTHESIS
Three major
feedback mechanisms cooperate in regulating the overall rate of de novo purine
nucleotide synthesis and the relative rates of formation of the two end
products, adenylate and guanylate. The first mechanism is exerted on the first
reaction that is unique to purine synthesis — transfer of an amino group to
PRPP to form 5-phosphoribosylamine. This reaction is catalyzed by the
allosteric enzyme glutamine-PRPP amidotransferase, which is inhibited by the
end products IMP, AMP, and GMP. AMP and GMP act synergistically in this
concerted inhibition. Thus, whenever either AMP or GMP accumulates to excess,
the first step in its biosynthesis from PRPP is partially inhibited.
In the second
control mechanism, exerted at a later stage, an excess of GMP in the cell
inhibits formation of xanthylate from inosinate by IMP dehydrogenase, without
affecting the formation of AMP. Conversely, an accumulation of adenylate
inhibits formation of adenylosuccinate by adenylosuccinate synthetase, without
affecting the biosynthesis of GMP. In the third mechanism, GTP is required in
the conversion of IMP to AMP ( step 1 ), whereas ATP is required for conversion
of IMP to GMP (step 4 ), a reciprocal arrangement that tends to balance the
synthesis of the two ribonucleotides.
The final
control mechanism is the inhibition of PRPP synthesis by the allosteric
regulation of ribose phosphate pyrophosphokinase. This enzyme is inhibited by
ADP and GDP, in addition to metabolites from other pathways of which PRPP is a
starting point.
Biosynthesis of NAD+
Nicotinamide adenine dinucleotide (NAD+) and its
phosphorylated analog, NADP+, are important coenzymes that
participate in a number of biological processes involving electron transfer.
NAD+ contains an AMP moiety as part of the molecule:
NAD+ synthesis requires nicotinate (vitamin B6),
which is derived from tryptophan. In the first step, nicotinate ribonucleotide
is formed from nicotinate and PRPP:
In the following steps, an AMP moiety is transferred from ATP to
nicotinate ribonucleotide to form desamido-NAD+. Finally, the
carboxyl group of desamido-NAD is converted to amide using glutamine as an
ammonia donor:
NADP is obtained by phosphorylation of the 2'-OH of the adenine ribose
by ATP in the presence of NAD+ kinase.
The essential rate limiting steps in purine
biosynthesis occur at the first two steps of the pathway. The synthesis of PRPP
by PRPP synthetase is feed-back inhibited by purine-5'-nucleotides
(predominantly AMP and GMP). Combinatorial effects of those two nucleotides are
greatest, e.g., inhibition is maximal when the correct concentration of both
adenine and guanine nucleotides is achieved.
The amidotransferase reaction catalyzed by PRPP
amidotransferase is also feed-back inhibited allosterically by binding ATP, ADP
and AMP at one inhibitory site and GTP, GDP and GMP at another. Conversely the
activity of the enzyme is stimulated by PRPP.
Additionally, purine biosynthesis is regulated in
the branch pathways from IMP to AMP and GMP. The accumulation of excess ATP
leads to accelerated synthesis of GMP, and excess GTP leads to accelerated
synthesis of AMP.
Catabolism
and Salvage of Purine Nucleotides
Catabolism of the purine nucleotides leads ultimately to the production
of uric acid which is insoluble and is excreted in the urine as sodium urate
crystals.
Catabolism of
Purine Nucleotides
The synthesis of nucleotides from the purine
bases and purine nucleosides takes place in a series of steps known as the
salvage pathways. The free purine bases, adenine, guanine, and hypoxanthine,
can be reconverted to their corresponding nucleotides by phosphoribosylation.
Two key transferase enzymes are involved in the salvage of purines: adenosine phosphoribosyltransferase
(APRT), which catalyzes the following reaction:
adenine + PRPP <----> AMP + PPi
and
hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the
following reactions:
hypoxanthine + PRPP <----> IMP
+ PPi
guanine + PRPP <----> GMP + PPi
A critically important enzyme of purine salvage
in rapidly dividing cells is adenosine deaminase (ADA) which catalyzes the
deamination of adenosine to inosine. Deficiency in
Salvage pathways for purine
nucleotides
Purine nucleotide phosphorylases (PNPs) can also contribute to the salvage
of the bases through a reversal of the catabolism pathways. However, this
pathway is less significant than those catalyzed by the
phosphoribosyltransferases.
The synthesis of AMP from IMP and the salvage of IMP via AMP catabolism
have the net effect of deaminating aspartate to fumarate. This process has been
termed the purine nucleotide cycle (see diagram below). This cycle is very
important in muscle cells. Increases in muscle activity create a demand for an
increase in the TCA cycle,
in order to generate more NADH for the production of ATP. However, muscle lacks
most of the enzymes of the major anapleurotic reactions. Muscle replenishes
TCA-cycle intermediates in the form of fumarate generated by the purine
nucleotide cycle.
The purine nucleotide cycle serves an important function within
exercising muscle. The generation of fumarate provides skeletal muscle with
its' only source of anapleurotic substrate for the TCA cycle.
In order for continued operation of the cycle during exercise, muscle protein
must be utilized to supply the amino nitrogen for the generation of aspartate.
The generation of asparate occurs by the standard transamination reactions that
interconvert amino acids with α-ketoglutarate to form glutamate and glutamate with
oxaloacetate to form aspartate. Myoadenylate deaminase is the muscle-specific
isoenzyme of AMP deaminase, and deficiencies in myoadenylate deaminase lead to
post-exercise fatigue, cramping and myalgias.
Clinical
Significances of Purine Metabolism
Clinical problems associated with nucleotide metabolism in humans are
predominantly the result of abnormal catabolism of the purines. The clinical
consequences of abnormal purine metabolism range from mild to severe and even
fatal disorders. Clinical manifestations of abnormal purine catabolism arise
from the insolubility of the degradation byproduct, uric acid.
Nucleotides
to Bases
Guanine nucleotides are hydrolyzed to the
nucleoside guanosine which undergoes phosphorolysis to guanine
and ribose 1-P. Man's intracellular nucleotidases are not very active
toward AMP, however. Rather, AMP is deaminated by the enzyme adenylate (AMP)
deaminase to IMP. In the catobilsm of purine nucleotides, IMP is further
degraded by hydrolysis with nucleotidase to inosine and then
phosphorolysis to hypoxanthine.
Adenosine does occur but usually arises from
S-Adenosylmethionine during the course of transmethylation reactions. Adenosine
is deaminated to inosine by an adenosine deaminase. Deficiencies in either adenosine
deaminase or in the purine nucleoside phosphorylase lead to two
different immunodeficiency diseases by mechanisms that are not clearly understood.
With adenosine deaminase deficiency, both T and B-cell immunity is
affected. The phosphorylase deficiency affects the T cells but B cells
are normal. In September,
Whether or not methylated purines are catabolized depends upon
the location of the methyl group. If the methyl is on an -NH2, it is
removed along with the -NH2 and the core is metabolized in the usual
fashion. If the methyl is on a ring nitrogen, the compound is excreted
unchanged in the urine.
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.
URIC ACID
- 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).
URIC ACID METABOLISM:
- Sources of purines in
humans:
- Diet
- Degradation of endogenous
nucleotides
- De novo synthesis (energy
requiring process).
- Purines are degraded to
uric acid.
- Urate is excreted via 2
routes:
- 1/3: Secretion into the
gut, and subsequent degradation by bacterial uricase to CO2 and NH3.
- 2/3: Renal excretion:
- Urate is filtered at the
glomeruli.
- Proximal tubular
reabsorption of 99% of filtered load.
- More distal part of
proximal tubules: secretion (also some reabsorption, but less than secretion).
- Net excretion of 10% of
filtered load.
- Body urate pool (and
plasma concentration) depends on the relative rates of urate formation and
urate excretion.
-
- De novo synthesis leads to
the formation of IMP (inosine monophosphate), which can be converted to AMP
(adenosine monophosphate) and GMP (guanosine monophosphate) (NUCLEOTIDES:
purine base + sugar + PO4).
- Nucleotide degradation
involves the formation of the respective nucleosides (inosine, adenosine and
guanosine) (NUCLEOSIDES: purine base + sugar), these are subsequently
metabolised to the respective purine bases (hypoxanthine, adenine and guanine)
(PURINE BASES).
- Hypoxanthine and guanine
can be metabolised directly to xanthine, but AMP/adenosine have to be converted
to IMP/inosine first.
- Xanthine is metabolised to
uric acid by the enzyme xanthine oxidase, also responsible for conversion of
hypoxanthine to xanthine.
- Because de novo synthesis
is an energy requiring process, excretion of uric acid results in net energy
loss. However, salvage pathways exist to convert purines back to their parent
nucleotides and are therefore energy saving – accomplished by the following
enzymes:
- For guanine and
hypoxanthine: HGPRT (hypoxanthine-guanine phosphoribosyl transferase).
- For adenine: APRT (adenine
phosphoribosyl transferase).
DEFINITION AND CAUSES
Gout is a form of arthritis that occurs as a result of
the build-up of uric acid in the body and the joint fluid (hyperuricemia). This
accumulation of uric acid typically occurs when the body has difficulty
processing certain protein substances called purines (PURE-EENS) that are found
naturally in our diets.
The actual build-up of uric acid can result
when the body has difficulty eliminating uric acid through the kidneys and
urine, or in some cases, when the body produces too much uric acid. The
tendency to accumulate uric acid is often hereditary. It can, however, skip an
individual or even a generation and reappear in the children of someone who has
no signs of gout. While it is most commonly seen in males between fifty and
sixty years of age, gout does occur in females and in younger males.
SYMPTOMS
Gout usually starts with a sudden onset of intense
pain in one or more joints, usually the big toe joint of the foot. The pain is
accompanied by redness, swelling and warmth over the joint. Typically, the
patient does not recall injuring the joint before the pain started. Many
patients say they first noticed pain in the middle of the night or upon arising
in the morning.
While symptoms most commonly occur in the big
toe joint, any joint may be involved. Other common sites are the instep of the
foot, the ankle, or the knee. When the foot is involved, wearing shoes is
difficult and painful, as are attempts to move the joint or stand on the foot.
DIAGNOSIS
The diagnosis is based on a personal and family
history, as well as on the doctor's examination, which often finds the classic
signs of gout and makes the diagnosis clear. Blood tests often are performed to
determine uric acid levels, and the joint fluid is examined to look for uric
acid crystals. X-rays also may be performed to examine both the bones and
joints to rule out abnormal changes associated with gout.
GOUT:
- Gout
is a group of metabolic diseases associated with hyperuricaemia and deposition
of crystals of monosodium urate in tissues.
- Prevalence:
3/1000, males affected more than females (8-10:1).
- Presentation
usually occurs in males over 30 years of age and females after the menopause.
- There
are 4 stages in the development of the disorder:
1.
Asymptomatic hyperuricaemia:
- Hyperuricaemia
is usually present for many years before the onset of symptoms.
- NB:
Only
2. Acute gouty arthritis:
- Classical
presentation is acute inflammation of the metatarsophalangeal joint of the big
toe (70%), and the first attack is usually monoarticular (affects only 1
joint).
- Other
joints that may be involved are the ankle, knee, wrist, elbow, and small joints
of hands and feet.
3.
Intercritical gout:
- Some
patients may have only 1 attack, whilst others have recurrent attacks at
shorter intervals.
- Between
attacks the patient is usually asymptomatic except for hyperuricaemia.
4.
Chronic tophaceous gout:
- This
follows recurrent attacks and is characterised by the development of tophi
(swellings containing uric acid crystals) in the periarticular tissue.
- Other
sites include the helix of the ear, bursae and tendons.
- Complications
of hyperuricaemia:
- Urolithiasis
(kidney stones):
- 10%
of gouty patients develop urate stones and 10% of all renal calculi are due to
urate.
- Renal
failure:
- Acute
renal failure due to obstructive uropathy (urate crystals) may occur during
cytotoxic treatment of malignancy (allopurinol cover should be used), and has
also been described in gouty subjects after severe exercise.
- Progressive
chronic renal insufficiency is an important cause of morbidity and mortality in
untreated chronic tophaceous gout.
- Associated
conditions:
- Alcoholism
- Dysmetabolic
syndrome (Insulin resistance syndrome)(syndrome X): Obesity, characteristic
dyslipidaemia (increased triglycerides, decreased HDL cholesterol, small dense
LDL), hypertension, impaired glucose tolerance, prothrombotic state.
- Diagnosis:
- The
laboratory evaluation of hyperuricaemia is discussed below. It is important to
recognise that:
- Hyperuricaemia
is not synonymous with gout (
- Gout
can be precipitated by a sudden change (either increase or decrease) in urate
concentration.
- An
acute gout attack may be associated with a normal plasma urate level (due to a
fall in urate level as seen with a change in diet, decrease in alcohol
consumption), although hyperuricaemia will be demonstrated at some stage.
- Diagnosis
is therefore usually made on clinical grounds.
- Definitive
diagnosis: Examination of synovial fluid under polarizing light microscope for
monosodium urate crystals (needle shaped and strongly negatively birefringent).
- Therapeutic
agents used in gout and hyperuricaemia:
- Three
groups of drugs are available:
Allopurinol (structural analogue of hypoxanthine),
and its major metabolite, oxypurinol, inhibit the enzyme xanthine oxidase,
producing a decrease in the plasma and urinary concentrations of urate
(hypoxanthine does not accumulate if the salvage pathway is intact).
Initial treatment with allopurinol should be
covered with an anti-inflammatory agent, because an acute attack of gout can be
precipitated when the initial dose is given (sudden decrease in urate can cause
mobilisation from body pools).
2.
Uricosuric agents:
- These
drugs (eg Probenecid) increase the urinary excretion of urate by inhibiting
tubular reabsorption.
- 3. Anti-inflammatory agents:
- These
agents (eg colchicine and indomethacin) are used symptomatically to relieve the
pain of acute gouty arthritis.
- They
have no effect on plasma urate levels.
TREATMENT
The treatment of gout starts with
establishing the correct diagnosis. Oral anti-inflammatory medications are most
often used to manage the acute attack. While over the counter drugs may reduce
symptoms, they are rarely strong enough to treat the acute pain, swelling and
inflammation. If the gout attack is in the toe, it will typically help to
elevate the foot, avoid standing and walking, and wear only a loose slipper
until the individual can be seen by a podiatric surgeon.
Gout often can be controlled with proper
medication, both when there is an attack and on a long-term basis. It is
important that your doctor establish which of the two primary causes (producing
too much uric acid or not eliminating it properly) is involved in order to
treat the gout with the appropriate medication.
If gout attacks continue despite medical
treatment, if there are excessive deposits of gouty crystals within a joint, or
if arthritis causes continual discomfort, surgical treatment may be necessary
to remove the crystals (tophi) and repair the joint. Failure to consider
surgery when it is needed may result in permanent arthritis of the joint(s).
PREVENTION
Certain foods that are high in purines can
increase uric acid levels and thus bring on an acute attack of gout. These
foods include red meats, shellfish, beer, red wine and salt. Some medications,
such as diuretics (water pills) that are often used to control high blood
pressure or reduce swelling, also may cause an acute attack of gout. Stress,
infection, and trauma also are possible causes.
Drinking 6-8 glasses of water each day,
eating an appropriate diet, and evaluating current medications will reduce the
likelihood of an attack or lessen the severity should it occur. If you have a
personal or family history of gout, regular examinations by a podiatric surgeon
also will reduce the potential for an attack.
The inflammatory response is due to the crystals engaging the
caspase-1-activating inflammasome resulting in the production of interleukin-1β (IL-1β) and IL-18.
Most forms of gout are the result of excess purine production and consequent
catabolism or to a partial deficiency in the salvage enzyme, HGPRT. Most forms
of gout can be treated by administering the antimetabolite: allopurinol.
This compound is a structural analog of hypoxanthine that strongly inhibits
xanthine oxidase.
Two severe disorders,
both quite well described, are associated with defects in purine metabolism:
Lesch-Nyhan syndrome and severe combined immunodeficiency disease (SCID).
Lesch-Nyhan syndrome results from the loss of a functional HGPRT gene. The
disorder is inherited as a sex-linked trait, with the HGPRT gene on the X
chromosome (Xq26-q27.2). Patients with this defect exhibit not only severe
symptoms of gout but also a severe malfunction of the nervous system. In the
most serious cases, patients resort to self-mutilation. Death usually occurs
before patients reach their 20th year.
Introduction
to Lesch-Nyhan Syndrome
Lesch-Nyhan syndrome (LNS) is a disorder related to defects in the activity
of the purine nucleotide salvage enzyme, hypoxanthine-guanine
phosphoribosyltransferase (HGPRT). HGPRT catalyzes the following two
interconversions:
hypoxanthine + PRPP <---> IMP + PPi
guanine + PRPP <---> GMP + PPi
There are three over-lapping clinical syndromes associated with
deficiencies in HGPRT activity. Individuals that have less than 1.5% residual enzyme
activity exhibit debilitating neurologic disability, behavioral abnormalities
that include impulsive and self-mutilating behaviors and varying degrees of
cognitive disability in addition to overproduction of uric acid. This most
severe of the three clinical syndromes is Lesch-Nyhan syndrome. Patients 1.5%
to 8% of residual enzyme activity exhibit neurologic disability that ranges
from clumsiness to debilitating pyramidal (CNS neurons involved in voluntary
motor movement) and extrapyramidal motor dysfunction in addition to
overproduction of uric acid. In cases where at least 8% of normal HGPRT
activity is present, patients exhibit overproduction of uric acid and
associated hyperuricemia, renal lithiasis (kidney stones) and gout.
The latter circumstance (partial deficiency with at least 8% enzyme activity)
is associated with Kelley-Steegmiller
syndrome.
Lesch-Nyhan syndrome is inherited as an X-linked recessive disorder with
an incidence of approximately 1:380,000. Since it is an X-linked disease it is
found almost exclusively in males although affected females have been
identified albeit very rarely.
The HGPRT gene (symbol = HPRT) is located on the X chromosome
(Xq26-q27.2) spanning 44 kbp and composed of 9 exons. In addition, 4
pseudogenes have been found. Over 270 different mutations in the HPRT gene have
been identified in LNS patients. Alterations to the gene include single base
insertions and deletions, large deletions, amino acid substitutions and stop
codon mutations.
Clinical Features of Lesch-Nyhan Syndrome
The characteristic clinical features of the
Lesch-Nyhan syndrome are mental retardation, spastic cerebral palsy,
choreoathetosis, uric acid urinary stones, and self-destructive biting of
fingers and lips. The overal clinical features of LNS can be divided into three
broad categories. These include uric acid overproduction and its associated
consequences (e.g. gouty arthritis and renal lithiasis), neurobehavioral
dysfunction indicative of central nervous system involvement, and growth
retardation. As indicated (and as expected from uric acid overproduction) LNS
patients manifest with many of the symptoms of classic gout and will not
be covered here.
All patients with Lesch-Nyhan syndrome manifest
with profound motor dysfunction that is recognizable within the first 3 to 9
months of life. Infants fail to develop the ability to hold up their heads or
to sit unaided. Further motor development will be delayed and the onset of
pyramidal and extrapyramidal signs become evident by 1 to 2 years of age. In
LNS patients there are three major signs of pyramidal dysfunction: spasticity,
hyperreflexia and the extensor plantar reflex (also known as the Babinski
reflex: the great toe flexes toward the top of the foot and the other toes fan
out after the sole of the foot has been firmly stroked). Extrapyramidal dysfunction
in LNS patients is primarily evident as dystonia (sustained muscle contractions
causing twisting and repetitive movements or abnormal postures) although many
patients also exhibit choreoatheosis (involuntary movement disorder in
association with slow continuous writhing particularly of the hands and feet).
Most commonly associated with Lesch-Nyhan
syndrome is the behavioral dysfunction manifest with impulsivity and
self-mutilation particularly of the lips, fingers and tongue. LNS patients will
often strike out at people around them, spit on people and use foul language.
These symptoms are analogous to the uncontrollable compulsions associated with
Tourette syndrome. The self-injury behavior is clearly an involuntary action as
most LNS patients will learn to call out for help when they feel the compulsive
behavior overtaking them, or they will sit on their hands or wear socks or
gloves to limit the self-injurious behavior. Although the precise cause of the
self-mutilating behavior in LNS patients is not clearly understood it is most
likely that it is a form of obsessive-compulsive disorder.
SCID is most often (90%) caused by a deficiency
in the enzyme adenosine deaminase (ADA). This is the enzyme responsible for
converting adenosine to inosine in the catabolism of the purines. This
deficiency selectively leads to a destruction of B and T lymphocytes, the cells
that mount immune responses. In the absence of ADA, deoxyadenosine is
phosphorylated to yield levels of dATP that are 50-fold higher than normal. The
levels are especially high in lymphocytes, which have abundant amounts of the
salvage enzymes, including nucleoside kinases. High concentrations of dATP
inhibit ribonucleotide reductase (see below), thereby preventing other dNTPs
from being produced. The net effect is to inhibit DNA synthesis. Since
lymphocytes must be able to proliferate dramatically in response to antigenic
challenge, the inability to synthesize DNA seriously impairs the immune
responses, and the disease is usually fatal in infancy unless special
protective measures are taken. A less severe immunodeficiency results when
there is a lack of purine nucleoside phosphorylase (PNP), another
purine-degradative enzyme.
Disorders of Purine Metabolism
|
Disorder |
Defect |
Nature of Defect |
Comments |
|
3
different enzyme defects can lead to gout: PRPP
synthetase HGPRTa glucose-6-phosphatase
|
elevated activity deficiency deficiency |
hyperuricemia |
|
|
HGPRT |
lack of enzyme |
see above |
|
|
ADAb |
lack of enzyme |
see above |
|
|
Immunodeficiency |
PNPc |
lack of enzyme |
see above |
|
APRTd |
lack of enzyme |
2,8-dihydroxyadenine, renal lithiasis |
|
|
Xanthine oxidase |
lack of enzyme |
hypouricemia
and xanthine renal lithiasis |
|
|
Glucose-6-phosphatase |
enzyme deficiency |
see above |
ahypoxanthine-guanine
phosphoribosyltransferase
badenosine deaminase
cpurine nucleotide phosphorylase
dadenosine phosphoribosyltransferase
INBORN ERRORS OF PURINE METABOLISM:
A. Hypoxanthine-guanine
phosphoribosyl transferase (HGPRT) deficiency (Lesch-Nyhan syndrome):
- The Lesch-Nyhan syndrome is
an X-linked recessive disorder, due to severe deficiency of HGPRT.
- It is characterised by
hyperuricaemia, mental deficiency, spasticity, choreoathetosis and
self-mutilation.
- Hyperuricaemia is due to
decreased activity of the salvage pathway causing decreased purine
reutilization and increased uric acid synthesis. Relatively low levels of
nucleotides result in decreased inhibition of de novo synthesis, resulting in
further overload of the non-functioning salvage pathway and increased uric acid
production.
B. Glucose 6-phosphatase
deficiency (Glycogen storage disease type I/ Von Gierke’s disease): (see figure
4)
- Deficiency of glucose
6-phosphatase (final enzyme in glycogenolysis pathway) results in accumulation
of glycogen, and hypoglycemia.
- Increased metabolism of
glucose 6-phosphate through glycolysis results in lactic acidosis.
- Increased metabolism of
glucose 6-phosphate through pentose phosphate pathway increases formation of
ribose 5-phosphate and NADPH.
- Ribose 5-phosphate is a
substrate for increased de novo purine nucleotide synthesis, which is
subsequently degraded to uric acid resulting in hyperuricaemia.
- NADPH is a coenzyme in
triglyceride synthesis, and overproduction results in hypertriglyceridaemia.
- Hyperuricaemia is
aggravated by increased lactic acid which inhibits renal excretion of uric
acid.
CAUSES OF HYPERURICAEMIA:
A. Physiological/environmental
factors
B. Primary hyperuricaemia
Overproduction:
- Idiopathic
- Glucose-6-phosphatase
deficiency (Von Gierke’s disease)
- HGPRT deficiency
(Lesch-Nyhan syndrome)
Reduced excretion:
- Idiopathic
C. Secondary hyperuricaemia
Overproduction:
- Increased nucleic acid
turnover:
- Myeloproliferative
disease, eg polycythemia vera
- Lymphoma, leukemia
- Multiple myeloma
- Cytotoxic therapy of
malignancies
- Psoriasis
- Disordered ATP metabolism:
- Alcohol (increased ATP
turnover)
- Tissue hypoxia
- Excessive dietary purine
intake
Reduced excretion:
- Decreased glomerular
filtration:
- Renal failure
- Decreased secretion (competition
with urate for tubular secretion):
- Lactic acidosis – alcohol,
exercise
- Ketoacidosis – alcohol,
diabetes, starvation
- Drugs – low dose
salicylate
- Increased reabsorption:
- Hypovolemia, eg diuretics.
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.
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-Acetylglutamate.
Synthesis of UMP from carbamoyl phosphate. Carbamoyl phosphate utilized
in pyrimidine nucleotide synthesis differs from that synthesized in the urea
cycle; it is synthesized from glutamine instead of ammonia and is synthesized
in the cytosol. The reaction is catalyzed by carbamoyl phosphate synthetase II
(CPS-II). Subsequently carbamoyl phosphate is incorporated into the pyrimidine
nucleotide biosynthesis pathway through the action of aspartate
transcarbamoylase, ATCase (enzyme #1) which is the rate limiting step in
pyrimidine biosynthesis. Following completion of UMP synthesis it can be
phosphorylated to UTP and utilized as a substrate for CTP synthase for the
synthesis of CTP. Uridine nucleotides are also the precursors for de novo
synthesis of the thymine nucleotides. Place mouse over green intermediate names
to see structure.
Enzyme
names:
1. aspartate transcarbamoylase, ATCase
2. carbamoyl aspartate dehydratase
3. dihydroorotate dehydrogenase
4. orotate phosphoribosyltransferase
5.
orotidine-5'-phosphate carboxylase
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.
Formation of the Nucleotides
Orotic acid is converted to its nucleotide with PRPP. 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. After conversion of
UMP to the triphosphate, the amide of glutamine is added, at the expense of
ATP, to yield CTP.
Control
The control of pyrimidine nucleotide synthesis in man is exerted
primarily at the level of cytoplasmic CPS II. UTP inhibits the
enzyme, competitively with ATP. PRPP activates it. Other secondary sites
of control also exist (e.g. OMP decarboxylase is inhibited by UMP and CMP).
These are probably not very important under normal circumstances.
In bacteria, aspartate transcarbamylase is the control enzyme. There is
only one carbamoyl phosphate synthetase in bacteria since they do not have
mitochondria. Carbamoyl phosphate, thus, participates in a branched pathway in
these organisms that leads to either pyrimidine nucleotides or arginine.
Orotic aciduria refers to an excessive excretion of orotic
acid in urine. It causes a characteristic form of anemia and may be associated
with mental and physical retardation.
In addition to the characteristic excessive orotic
acid in the urine, patients typically have megaloblastic anemia which cannot be
cured by administration of vitamin B12 or folic acid.
It also can cause inhibition of RNA and DNA
synthesis and failure to thrive. This can lead to mental and physical
retardation.
Its hereditary form, an autosomal recessive
disorder, can be caused by a deficiency in the enzyme UMPS, a bifunctional
protein that includes the enzyme activities of orotate
phosphoribosyltransferase and orotidine 5'-phosphate decarboxylase.
It can also arise secondary to blockage of the urea
cycle, particularly in ornithine transcarbamylase deficiency (or OTC
deficiency). You can distinguish this increase in orotic acid secondary to OTC
deficiency from hereditary orotic aciduria (seen above) by looking at blood
ammonia levels and the BUN. In OTC deficiency, because the urea cycle backs up,
you will see hyperammonemia and a decreased BUN.
Administration of cytidine monophosphate and uridine
monophosphate reduces urinary orotic acid and the anemia.
Administration of uridine, which is converted to
UMP, will bypass the metabolic block and provide the body with a source of
pyrimidine.
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.
Formation
of Deoxyribonucleotides
De novo synthesis
and most of the salvage pathways involve the ribonucleotides. (Exception is the
small amount of salvage of thymine indicated above.) Deoxyribonucleotides for
DNA synthesis are formed from the ribonucleotide diphosphates (in mammals and E.
coli).
A base diphosphate (BDP) is reduced at the 2'
position of the ribose portion using the protein, thioredoxin and the
enzyme nucleoside diphosphate reductase. Thioredoxin has two sulfhydryl
groups which are oxidized to a disulfide bond during the process. In order to
restore the thioredoxin to its reduced for so that it can be reused, thioredoxin
reductase and NADPH are required.
This system is very tightly controlled by a
variety of allosteric effectors. dATP is a general inhibitor for all substrates
and ATP an activator. Each substrate then has a specific positive effector (a
BTP or dBTP). The result is a maintenance of an appropriate balance of the
deoxynucleotides for DNA synthesis.
Synthesis of the Thymine Nucleotides
The de novo pathway to dTTP synthesis
first requires the use of dUMP from the metabolism of either UDP or CDP. The
dUMP is converted to dTMP by the action of thymidylate synthase. The methyl
group (recall that thymine is 5-methyl uracil) is donated by N5,N10-methylene
THF, similarly to the donation of methyl groups during the biosynthesis of the
purines. The unique property of the action of thymidylate synthase is that the
THF is converted to dihydrofolate (DHF), the only such reaction yielding DHF
from THF. In order for the thymidylate synthase reaction to continue, THF must
be regenerated from DHF. This is accomplished through the action of
dihydrofolate reductase (DHFR). THF is then converted to N5,N10-THF
via the action of serine hydroxymethyl transferase. The crucial role of DHFR in
thymidine nucleotide biosynthesis makes it an ideal target for chemotherapeutic
agents (see below).
Synthesis of
dTMP from dUMP
The salvage pathway to dTTP synthesis involves
the enzyme thymidine kinase which can use either thymidine or deoxyuridine as substrate:
thymidine + ATP <----> TMP +
ADP
deoxyuridine + ATP <----> dUMP
+ ADP
The activity of thymidine kinase (one of the
various deoxyribonucleotide kinases) is unique in that it fluctuates with the
cell cycle, rising to peak activity during the phase of DNA synthesis; it is
inhibited by dTTP.
DNA synthesis also requires dTMP (dTTP). This is not synthesized in the de
novo pathway and salvage is not adequate to maintain the necessary amount.
dTMP is generated from dUMP using the folate-dependent one-carbon pool.
Since the nucleoside diphosphate reductase is not
very active toward UDP, CDP is reduced to dCDP which is converted to dCMP. This
is then deaminated to form dUMP. In the presence of 5,10-Methylene
tetrahydrofolate and the enzyme thymidylate synthetase, the carbon
group is both transferred to the pyrimidine ring and further reduced to a
methyl group. The other product is dihydrofolate which is subsequently
reduced to the tetrahydrofolate by dihydrofolate reductase.
Clinical Relevance of Tetrahydrofolate
Tetrahydrofolate (THF) is regenerated from the dihydrofolate (DHF)
product of the thymidylate synthase reaction by the action of dihydrofolate
reductase (DHFR), an enzyme that requires NADPH. Cells that are unable to
regenerate THF suffer defective DNA synthesis and eventual death. For this
reason, as well as the fact that dTTP is utilized only in DNA, it is
therapeutically possible to target rapidly proliferating cells over
non-proliferating cells through the inhibition of thymidylate synthase. Many
anti-cancer drugs act directly to inhibit thymidylate synthase, or indirectly,
by inhibiting DHFR.
The class of molecules used to inhibit thymidylate synthase is called
the suicide substrates because they irreversibly inhibit the enzyme. Molecules
of this class include 5-fluorouracil and 5-fluorodeoxyuridine. Both are
converted within cells to 5-fluorodeoxyuridylate, FdUMP. It is this drug
metabolite that inhibits thymidylate synthase. Many DHFR inhibitors have been
synthesized, including methotrexate, aminopterin, and trimethoprim. Each
of these is an analog of folic acid.
Regulation of Pyrimidine Biosynthesis
The regulation of pyrimidine synthesis occurs
mainly at the first step which is catalyzed by aspartate transcarbamoylase,
ATCase. Inhibited by CTP and activated by ATP, ATCase is a multifunctional
protein in mammalian cells. It is capable of catalyzing the formation of
carbamoyl phosphate, carbamoyl aspartate, and dihydroorotate. The carbamoyl
synthetase activity of this complex is termed carbamoyl phosphate synthetase II
(CPS-II) as opposed to CPS-I, which is involved in the urea cycle.
ATCase, and therefore the activity of CPS-II, is
localized to the cytoplasm and prefers glutamine as a substrate. CPS-I of the
urea cycle is localized in the mitochondria and utilizes ammonia. The CPS-II
domain is activated by ATP and inhibited by UDP, UTP, dUTP, and CTP.
The role of glycine in ATCase regulation is to
act as a competitive inhibitor of the glutamine binding site. As in the
regulation of purine synthesis, ATP levels also regulate pyrimidine
biosynthesis at the level of PRPP formation. An increase in the level of PRPP
results in an activation of pyrimidine synthesis.
There is also regulation of OMP decarboxylase:
this enzyme is competitively inhibited by UMP and, to a lesser degree, by CMP.
Finally, CTP synthase is feedback-inhibited by CTP and activated by GTP.
Chemotherapeutic Agents
Thymidylate synthetase is particularly sensitive to availability of the
folate one-carbon pool. Some of the cancer chemotherapeutic agents interfere
with this process as well as with the steps in purine nucleotide synthesis
involving the pool.
Cancer chemotherapeutic agents like methotrexate
(4-amino, 10-methyl folic acid) and aminopterin (4-amino, folic
acid) are structural analogs of folic acid and inhibit dihydrofolate reductase.
This interferes with maintenance of the folate pool and thus of de novo
synthesis of purine nucleotides and of dTMP synthesis. Such agents are highly
toxic and administered under careful control.
Catabolism and Salvage of Pyrimidine Nucleotides
Catabolism of the pyrimidine nucleotides leads ultimately to β-alanine (when
CMP and UMP are degraded) or β-aminoisobutyrate (when dTMP is degraded) and NH3
and CO2. The β-alanine
and β-aminoisobutyrate
serve as -NH2 donors in transamination of α-ketoglutarate
to glutamate. A subsequent reaction converts the products to malonyl-CoA (which
can be diverted to fatty acid synthesis) or methylmalonyl-CoA (which is
converted to succinyl-CoA and can be shunted to the TCA cycle).
The salvage of pyrimidine bases has less clinical significance than that
of the purines, owing to the solubility of the by-products of pyrimidine
catabolism. However, as indicated above, the salvage pathway to thymidine
nucleotide synthesis is especially important in the preparation for cell
division. Uracil can be salvaged to form UMP through the concerted action of
uridine phosphorylase and uridine kinase, as indicated:
uracil + ribose-1-phosphate <----> uridine + Pi
uridine + ATP ----> UMP + ADP
Deoxyuridine is also a substrate for uridine phosphorylase. Formation of
dTMP, by salvage of dTMP requires thymine phosphorylase and the previously
encountered thymidine kinase:
thymine + deoxyribose-1-phosphate <---->
thymidine + Pi
thymidine + ATP ----> dTMP + ADP
The salvage of deoxycytidine is catalyzed by
deoxycytidine kinase:
deoxycytidine + ATP <----> dCMP + ADP
Deoxyadenosine and deoxyguanosine are also substrates for deoxycytidine
kinase, although the Km for these substrates is much higher than for
deoxycytidine.
The major function of the pyrimidine nucleoside kinases is to maintain a
cellular balance between the level of pyrimidine nucleosides and pyrimidine
nucleoside monophosphates. However, since the overall cellular and plasma
concentrations of the pyrimidine nucleosides, as well as those of
ribose-1-phosphate, are low, the salvage of pyrimidines by these kinases is
relatively inefficient.
Clinical Significances of Pyrimidine Metabolism
Because the products of pyrimidine catabolism are
soluble, few disorders result from excess levels of their synthesis or
catabolism. Two inherited disorders affecting pyrimidine biosynthesis are the
result of deficiencies in the bifunctional enzyme catalyzing the last two steps
of UMP synthesis, orotate phosphoribosyl transferase and OMP decarboxylase.
These deficiencies result in orotic aciduria
that causes retarded growth, and severe anemia caused by hypochromic
erythrocytes and megaloblastic bone marrow. Leukopenia is also common in orotic
acidurias. The disorders can be treated with uridine and/or cytidine, which
leads to increased UMP production via the action of nucleoside kinases. The UMP
then inhibits CPS-II, thus attenuating orotic acid production.
Disorders of Pyrimidine Metabolism
|
Disorder |
Defective Enzyme |
Comments |
|
orotate phosphoribosyl transferase and OMP
decarboxylase |
see above |
|
|
OMP decarboxylase |
see above |
|
|
Orotic aciduria due to OTC deficiency (no hematologic component) |
the urea cycle enzyme, ornithine
transcarbamoylase, is deficient |
increased mitochondrial carbamoyl phosphate exits
and augments pyrimidine biosynthesis; hepatic encephalopathy |
|
β-aminoisobutyric
aciduria |
transaminase, affects urea cycle function during
deamination of α-amino
acids to α-keto acids |
benign,
frequent in Orientals |
|
drug
induced orotic aciduria |
OMP decarboxylase |
allopurinol and 6-azauridine treatments cause
orotic acidurias without a hematologic component; their catabolic by-products
inhibit OMP decarboxylase |
Formation of Deoxyribonucleotides
The typical cell contains 5 to10 times as much RNA (mRNAs, rRNAs and
tRNAs) as DNA. Therefore, the majority of nucleotide biosynthesis has as its
purpose the production of rNTPs. However, because proliferating cells need to
replicate their genomes, the production of dNTPs is also necessary. This
process begins with the reduction of rNDPs, followed by phosphorylation to
yield the dNTPs. The phosphorylation of dNDPs to dNTPs is catalyzed by the same
nucleoside diphosphate kinases that phosphorylates rNDPs to rNTPs, using ATP as
the phosphate donor.
Ribonucleotide reductase (RR) is a multifunctional enzyme that contains
redox-active thiol groups for the transfer of electrons during the reduction
reactions. In the process of reducing the rNDP to a dNDP, RR becomes oxidized.
RR is reduced in turn, by either thioredoxin or glutaredoxin. The ultimate
source of the electrons is NADPH. The electrons are shuttled through a complex
series of steps involving enzymes that regenerate the reduced forms of
thioredoxin or glutaredoxin. These enzymes are thioredoxin
reductase and glutathione reductase respectively.
Ribonucleotide reductase reactions
Ribonucleotide reductase is the only enzyme used in
the generation of all the deoxyribonucleotides. Therefore, its activity and
substrate specificity must be tightly regulated to ensure balanced production
of all four of the dNTPs required for DNA replication. Such regulation occurs
by binding of nucleoside triphosphate effectors to either the activity sites or
the specificity sites of the enzyme complex. The activity sites bind either ATP
or dATP with low affinity, whereas the specificity sites bind ATP, dATP, dGTP,
or dTTP with high affinity. The binding of ATP at activity sites leads to
increased enzyme activity, while the binding of dATP inhibits the enzyme. The
binding of nucleotides at specificity sites effectively allows the enzyme to
detect the relative abundance of the four dNTPs and to adjust its affinity for
the less abundant dNTPs, in order to achieve a balance of production.
thioredoxin reductase and glutathione reductase respectively.
Interconversion of the
Nucleotides
During the catabolism of nucleic acids, nucleoside
mono- and diphosphates are released. The nucleosides do not accumulate to any
significant degree, owing to the action of nucleoside kinases. These include
both nucleoside monophosphate (NMP) kinases and nucleoside diphosphate (NDP)
kinases. The NMP kinases catalyze ATP-dependent reactions of the type:
(d)NMP + ATP <----> (d)NDP +
ADP
There
are four classes of NMP kinases that catalyze, respectively, the
phosphorylation of:
1. AMP and
dAMP; this kinase is known as adenylate kinase.
2. GMP and dGMP.
3. CMP, UMP and
dCMP.
4. dTMP.
The enzyme adenylate kinase is important for
ensuring adequate levels of energy in cells such as liver and muscle. The
predominant reaction catalyzed by adenylate kinase is:
2ADP <----> AMP + ATP
The
NDP kinases catalyze reaction of the type:
N1TP + N2DP
<----> N1DP + N2TP
N1 can represent a purine ribo- or
deoxyribonucleotide; N2 a pyrimidine ribo- or deoxyribonucleotide.
The activity of the NDP kinases can range from 10 to 100 times higher than that
of the NMP kinases. This difference in activity maintains a relatively high
intracellular level of (d)NTPs relative to that of (d)NDPs. Unlike the
substrate specificity seen for the NMP kinases, the NDP kinases recognize a
wide spectrum of (d)NDPs and (d)NTPs.