Nucleotides, Folic Acid, Diseases and Meds Flashcards
Role of Nucleotides
Nucleotides are critically important cellular constituents. They serve as the energy “currency” of the cell, as “second messengers” in signal transduction cascades and as building blocks for DNA and RNA.
Purine Bases
- purine
- adenine
- guanine
- hypoxanthine
- xanthine
[…], a product of the pentose phosphate pathway is the major precursor for purine biosynthesis.
Ribose-5-phosphate, a product of the pentose phosphate pathway is the major precursor for purine biosynthesis
The first purine nucleotide (base + sugar + phosphate) produced is […].
The first purine nucleotide (base + sugar + phosphate) produced is inosinic acid (IMP).
Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP) is catalyzed by […]. PRPP is used in the synthesis of both purine and pyrimidine nucleotides by providing the ribose sugar and the alpha-phosphate. The subsequent reaction in purine biosynthesis is catalyzed by[…]. The final reaction requires […] with […] as co-factor.
Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP) is catalyzed by PRPP synthetase. PRPP is used in the synthesis of both purine and pyrimidine nucleotides by providing the ribose sugar and the alpha-phosphate. The subsequent reaction in purine biosynthesis is catalyzed by PRPP amidotransferase. The final reaction requires N10-formyl THF with amino acids Gly + Gln + Asp.
Purine Synthesis
- Adenosine monophosphate (AMP) and guanine monophosphate (GMP) are synthesized from inosine monophosphate via separate pathways.
- Synthesis of GMP requires nitrogen from gln and is inhibited by drugs, mycophenolate and ribavirin, which decrease activity of IMP dehydrogenase.
- AMP acquires nitrogen from asp.
- The monophosphate products are phosphorylated to the diphosphate and triphosphate forms.
- The end result of the purine synthesis pathway is the production of the purine ribonucleotides.
- Formation of purine deoxyribonucleotides is catalyzed by ribonucleotide reductase.
Allosteric inhibition of purine biosynthesis; […] and […] inhibit PRPP Synthetase and PRPP aminotransferase. In addition to this allosteric inhibition, […] allosterically stimulates formation of […] and […] allosterically stimulates the formation of […].
Allosteric inhibition of purine biosynthesis; GMP and AMP inhibit PRPP Synthetase and PRPP aminotransferase. In addition to this allosteric inhibition, ATP allosterically stimulates formation of GMP and GTP allosterically stimulates the formation of AMP.
Purine Nucleotide Degradation
- Degradation of purine nucleotides commences with the removal of phosphate from the nucleotide form, yielding a nucleoside (base + sugar).
- Adenosine is first converted to inosine by adenosine deaminase.
- The sugar group is removed by purine nucleoside phosphorylase to produce the purine bases guanine and hypoxanthine, as well as ribose-1- phosphate.
- The base products are converted to xanthine, which is oxidized by xanthine oxidase to uric acid. (Note that xanthine oxidase also converts hypoxanthine to xanthine.)
Uric Acid
- Uric acid, the purine degradative product, is a weak acid with a 5.8 pK [pH at which it is 50% ionized].
- Urate, the ionized form, is more water-soluble than is the protonated form.
-When urine is at a pH of 6.8, the molecule is 90% ionized and is 10-times more soluble than when urine is at pH 4.8 with only 10% ionized.
•Since the pH of urine is normally below 5.8, overproduction of uric acid can lead to formation of stones in the urinary collection system.
Purine Salvage Pathway
- Besides de novo synthesis, purine nucleotides also can be formed directly from the purine bases via a salvage pathway.
- The primary enzyme of the salvage pathway is hypoxanthineguanine phosphoribosyl transferase.
- HGPRT attaches PRPP, derived from the PRPP synthetase reaction, either to hypoxanthine to regenerate IMP or to guanine to restore GMP.
- Thus the salvage pathway prevents the irreversible destruction of hypoxanthine, guanine and adenine. Instead these purine bases can be reutilized.
- Because of the high-energy demand of the de novo synthesis pathway, the salvage pathway is an energy saving process.
- This pathway is also important for the salvage of dietary nucleotides. By salvaging the purine bases, production of uric acid is kept low, which is necessary for preventing gout.
- The salvage pathway is a critical source of purine nucleotides.
Disorders Asociated with Defects of Enzymes in the Purine Metabolic Pathway
•Hyperuricemia
-Type I Glycogen Storage Disease —> Gout
•Gout
*primary
*secondary
•Lesch-Nyhan Syndrome
Hyperuricemia - Type I Glycogen Storage Disease
- A defect of glucose-6-phosphatase in this disease increases the conversion of glucose-6-phosphate to ribose-5-phosphate via the pentose phosphate pathway.
- This in turn elevates production of purines through saturation of PRPP synthetase.
- Additionally, lactic acidosis associated with this disease can lower the pH of urine and thereby diminish excretion of uric acid as well.
Hyperuricemia - Gout
- Hyperuricemia may lead to gout, which describes clinically the physiological consequences associated with the excessive accumulation of uric acid in body fluids.
- The most common symptom of gout is arthritic pain in joints, which is caused by the deposition of urate crystals in cartilage surrounding the joint.
- Urate crystals may also develop into kidney stones.
- Gout occurs more commonly in men than in women, and even more rarely in premenopausal women. The overall prevalence of gout is about 1.4% of the population with an incidence of perhaps 7% in men over age 65.
Hyperuricemia - Gout Primary
- Primary gout is an inherited disorder
- Three different defects of PRPP synthetase have been identified.
- A superactive variant of this enzyme is associated with an increased Vmax
- An increased affinity (low Km) for ribose-5-phosphate thus leading to overproduction of PRPP.
- The loss of feedback inhibition of this enzyme by purine nucleotides. Consequently, when purine nucleotides reach an excessive concentration, there is no signal for shutting off their further production.
•Moderate defects of HGPRT that allow for at least 50% of normal activity lead to gout caused by overproduction of uric acid because of the inability to salvage the purine bases from complete degradation.
Hyperuricemia - Gout Primary
Hyperuricemia - Gout Primary
Pharmacological Treatment of Gout I
•allopurinol
- a competitive inhibitor of xanthine oxidase
- Hypoxanthine and xanthine are excreted during allopurinol therapy since xanthine oxidase uses both of these purines as substrates
- purine nucleotide synthesis is lowered during allopurinol treatment.
- Reducing the formation of uric acid by allopurinol treatment relieves the symptoms and decreases the possibility that uric acid kidney stones will form.
•febuxostat
- also a xanthine oxidase inhibitor, appears to be more effective than allopurinol in preventing acute attacks and reducing the size of the tophi deposits
- because this drug is not metabolized by kidney, it may be a better choice of drugs in patients with kidney disease
•probenecid and sulfinpyrazone
- lower the blood concentration of uric acid by increasing its excretion through inhibition of uric acid reabsorption
- taken with large amount of fluids to increase rapid excretion of uric acid and thereby lessen the risk of renal stones
***azathioprine and 6 mercaptopurine are antineoplastic drugs used block the synthesis of nucleotides, affecting the synthesis of both RNA and DNA leading to decreased proliferation of cancer cells***
Hyperuricemia - Lesch Nyhan Syndrome
- Considerable overproduction of uric acid occurs in individuals diagnosed with Lesch-Nyhan syndrome due to a severe defect in the gene encoding for HGPRT.
- The amount of HGPRT activity in these patients is <5%.
- The disease is X-linked recessive and thus generally limited to males.
- Lesch-Nyhan syndrome has a very early age of onset.
- The syndrome, in its most severe form is characterized by extremely aggressive behavior, which generally leads to self-mutilation, and by mental retardation.
- The severity of the consequences of the syndrome supports the important role for the salvage pathway.
- Most likely the enzyme has an essential role in non-hepatic tissues where de novo synthesis of purines occurs at a very slow rate. Therefore, non-hepatic tissues depend on circulating purine bases or nucleosides derived from the liver.
-Non-hepatic tissues are thought to take up the circulating purines and, through the action of HGPRT, form nucleotides.
- Though allopurinol is available for treatment, the overproduction of uric acid is so severe that there is little benefit from its use.
- Patients usually do not survive beyond their early teens, though there are exceptions.
Pyrimidine Synthesis
- The pyrimidines, like the purines, are synthesized in a multi-step pathway.
- The first step of the pathway is catalyzed by carbamoyl phosphate synthetase II, a cytoplasmic enzyme. This enzyme uses glutamine as a nitrogen donor.
-The product of this reaction, carbamoyl phosphate, is identical chemically to the carbamoyl phosphate made in the mitochondria by carbamoyl phosphate synthetase I as part of the urea cycle.
•Pyrimidine biosynthesis continues with the addition of aspartate, via aspartate transcarbamoylase, to provide the remainder of the ring elements.
-Aspartate transcarbamoylase is highly regulated in bacteria but not in humans.
•The final product of pyrimidine synthesis is uridine monophosphate (UMP). This can be phosphorylated to the triphosphate form (UTP), which in turn is converted into cytidine triphosphate (CTP).
-Both of these pyrimidines along with ATP and GTP are required for synthesis of RNA.
•Formation of pyrimidine deoxyribonucleotides is catalyzed by ribonucleotide reductase.
Disorders Associated with Defects of Enzymes in the Pyrimidine Metabolic Pathways
•Orotic Aciduria
-Orotic aciduria, an inherited autosomal recessive disorder of pyrimidine metabolism, is caused by a deficiency of the enzyme complex that includes both the orotate phosphoribosyl and orotidylic acid decarboxylase activities (Figure 8). As a result of this defect, orotic acid cannot be metabolized to UMP. Instead orotic acid accumulates in cells, and the body excretes it in large amounts. The pyrimidine pathway is diminished thereby limiting the de novo synthesis of uridine and cytidine nucleotides. Because of the nucleotide pool imbalance (purines favored over pyrimidines), children exhibit a failure to thrive. The disease is treated by administration of oral uridine that can be converted to UMP to bypass the enzymatic defect.
Formation of Deoxyribonucleotides
- The deoxyribonucleotides, which are needed for DNA formation (except thymidine) are synthesized directly from their respective ribonucleotides.
- The 2-hydroxyl group on the ribonucleotides is reduced to form the deoxyribonucleotide in a reaction catalyzed by ribonucleotide reductase.
- This reductase requires a reducing source, which a small protein is called thioredoxin.
- During the reduction of the ribonucleotide, thioredoxin is oxidized and must be reduced back to its active form by the action of thioredoxin reductase in a reaction that requires NADPH.
- The production of deoxyribonucleotides is tightly regulated to provide sufficient amounts of substrates for DNA synthesis during cell division and this occurs by feedback regulation via the deoxynucleotide triphosphates, especially dATP and dGTP.
Formation of Thymidine
- Thymidine nucleotides are required for DNA synthesis in place of UTP that is required for RNA formation.
- TTP is derived from TMP, which is formed from dUMP in a reaction catalyzed by thymidylate synthase.
- DeoxyUMP acquires a carbon from N5 ,N10 - methylenetetrahydrofolate, which is converted to dihydrofolate (DHF)
Metabolism of Folic Acid
- Folate (or folic acid) is a complex B water-soluble vitamin (B9).
- The dietary form undergoes activation and conversion to various forms that are used in several biochemical reactions.
- Folate serves as a donor of one-carbon groups.
- Ingested folate is first converted to dihydrofolate, which is reduced to tetrahydrofolate (THF) by dihydrofolate reductase.
- THF is the “backbone” for production of the other “active” forms of folate.
- N5 ,N10 -methylene THF gains a carbon from the side chain of serine, whose structure then becomes glycine (R group = hydrogen).
- N5 ,N10 -methylene THF is required for the synthesis of TMP and is a precursor for formation of two other folate derivatives.
- N5 ,N10 -methenyl THF is converted to N10 - formyl THF that is used in purine biosynthesis
- In the processing of cobalamin (vitamin B12), N5 -methyl THF provides a methyl group for the formation of methyl cobalamin.
*As a consequence of vitamin B12 deficiency, folate is trapped as N5 -methyl THF because the reaction from N5 ,N10 -methylene THF is not reversible.
*Under conditions of B12 deficiency, measurement of total folate can be misleading since N5 -methyl THF form is useless in the absence of cobalamin.
Inhibition of IMP Formation
•Mycophenolate
- IMP dehyrogenase
- Immunosuppressant to prevent transplant rejection
•Ribavirin
- IMP dehydrogenase
- Antiviral that stops viral RNA synthesis
ADA Deficiency
- In patients with adenosine deaminase (ADA) deficiency, both ATP and dATP accumulate in the cell because their degradation requires removal of the amino group from the adenine ring.
- Deficiency of this enzyme is an autosomal recessive disorder.
- Accumulated dATP inhibits the ribonucleotide reductase reaction creating an imbalance in the pool of deoxynucleotides leading to decreased synthesis of DNA.
- ADA deficiency leads to immunodeficiency because decreased synthesis of DNA causes a decline in the lymphocyte count.
- Deficiency of ADA is considered to be one of the major causes of severe combined immunodeficiency disease (SCID).
- SCID leaves the patients especially vulnerable to infectious diseases requiring them to be kept isolated in a sterile environment. SCID can be so severe that in many instances the immune system is virtually absent.
Inhibition of DNA Formation - Hydroxyurea
- Hydroxyurea is used as an antineoplastic drug in the treatment of several types of cancer including chronic myelogenous leukemia (CLL) and squamous cell carcinoma.
- Its mechanism of action as a chemotherapeutic agent most likely involves inhibition of ribonucleotide reductase thereby decreasing the availability of deoxynucleotides for DNA synthesis.
- Hydroxyurea is also used in the treatment of sickle cell disease to increase the amount of fetal hemoglobin that is unaffected by the mutation.
-The drug reduces the complications of patients with a history of severe symptoms.
- Side effects of the drug include nausea, vomiting, and mouth sores.
- More significantly it decreases bone marrow function that may reduce the number of red cells, white cells and platelets (pancytopenia).
Inhibition of DNA Formation - 5-Fluorouracil
- 5-Fluorouracil (5-FU) is used as a chemotherapeutic agent.
- It is referred to as a suicide inhibitor because the cell converts it to fluorodeoxyuridylate (F-dUMP) via orotate phosphoribosyl transferase.
- F-dUMP is a competitive inhibitor of thymidylate synthase.
- The drug is trademarked as Adrucil®, Carac®, or Efudex®.
- The most common side effects are nausea, vomiting, diarrhea, heartburn, and mouth sores.
Inhibition of DNA Formation - Methotrexate
- Methotrexate acts as a chemotherapeutic agent by inhibiting dihydrofolate reductase resulting in decreased synthesis of tetrahydrofolate (THF), which is a precursor for N5 ,N10 - methylene THF, a cofactor for thymidylate synthase.
- Thus this inhibitor ultimately disrupts DNA synthesis in cancer cells.
- The topical drug is used in the treatment of skin cancer.
- To protect normal cells against the harmful side effects of this drug, patients may be given leucovorin (folinic acid), a folic acid analog as a rescue treatment.
- Methotrexate is also used for treating rheumatoid arthritis, Crohn’s disease and psoriasis, reducing the active immune response in all these diseases.
- Trimethoprim and pyrimethamine also inhibit dihydrofolate reductase but in bacteria not in humans and thus act as antibiotics.
Inhibition of DNA Formation - 6-Mercaptopurine
- 6-Mercaptopurine (6-MP) blocks the synthesis of purine nucleotides by competing with hypoxanthine and guanine for HGPRT to reduce the salvage pathway.
- Thus 6-MP can affect the synthesis of both RNA and DNA leading to decreased proliferation of cancer cells. It has been used in the treatment of acute lymphocytic leukemia (ALL) and non-Hodgkins lymphoma, as well as Crohn’s disease and ulcerative colitis.
- Azathioprine is a prodrug that also inhibits purine formation through its conversion to 6-MP.
- Side effects include nausea, vomiting diarrhea, loss of appetite, and hair loss. More serious effects can include easy bruising, bleeding, and liver damage as indicated by abdominal pain and jaundice.
Pyrimidine Synthesis - Leflunomide
•Leflunomide (Arava®), used for treating rheumatoid arthritis decreases pyrimidine synthesis by inhibiting dihydroorotate dehydrogenase (dihydoorotic acid orotic acid)
