NUCLEIC ACID METABOLISM
BY SSEKATAWA KENNETH
NUCLEIC ACID METABOLISM: Ribonucleoside and deoxyribonucleoside phosphates (nucleotides) are essential for all cells.
Without them, neither DNA nor RNA can be produced and, therefore, proteins cannot be synthesized or cells cannot proliferate.
The purine and pyrimidine bases found in nucleotides can be synthesized de novo,
The ability to salvage nucleotides from sources within the body alleviates any nutritional requirement for nucleotides,
Therefore, 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.
Dietary nucleic acids: Ingested nucleic acids and nucleotides are dietarily nonessential
Extracellular hydrolysis of ingested nucleic acids ----- endonucleases, phosphodiesterases and nucleoside phosphorylase
Endonucleases degrade DNA and RNA to Mononucleotides / oligonucleotide
The mononucleotides may then be absorbed or converted to purine and pyrimidine bases.
Dietary nucleic acids: Oligonucleotides/mononucleotides are further digested by phosphodiesterases 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 or bases are not re-utilized
Purine bases are further degraded to uric acid
Pyrimidines to β-aminoiosobutyrate, NH3 and CO2.
Recap of nucleotide structure: Nucleotides are composed of: i. A nitrogenous base ii. A pentose sugar
i. One, two, or three phosphate groups (NMP, NDP &NTP) The nitrogen-containing bases belong to two families of compounds namely; i. The purines ii. The pyrimidines. Major purine and pyrimidine bases of nucleic acids. Some of the common names of these bases reflect the circumstances of their discovery. Guanine, for example, was first isolated from guano (bird manure), and thymine was first isolated from thymus tissue. Purine nucleotide biosynthesis: Purine nucleotides are synthesized in vivo at rates consistent with physiologic need.
Intracellular mechanisms sense and regulate the pool sizes of nucleotide triphosphates (NTPs)
The NTPs pool sizes rise during growth or tissue regeneration when cells are rapidly dividing.
Activation of Ribose-5-Phosphate
Both the salvage and de novo synthesis pathways of purine and pyrimidine biosynthesis lead to production of nucleoside-5'-phosphates (Nucleotides)
Through the utilization of an activated sugar intermediate and a class of enzymes called phosphoribosyltransferases.
The activated form of ribose-5-phosphate is 5- phosphoribosyl-1-pyrophosphate (PRPP)
Synthesis of PRPP: PRPP is an “activated pentose” It participates in the de novo synthesis and salvage of purines and pyrimidines. Synthesis of PRPP from ATP and ribose 5-phosphate is catalyzed by PRPP synthetase (ribose-phosphate pyrophosphokinase 1)
The enzyme is X-linked It is activated by inorganic phosphate and inhibited by purine nucleotides The sugar moiety of PRPP is ribose Therefore ribonucleotides are the end products of de novo purine synthesis.
Purine nucleotide biosynthesis (de novo pathway The atoms of the purine ring are contributed by a No of compounds, including amino acids (aspartic acid, glycine, and 10 glutamine), CO2, and N –formyltetrahydrofolate.
The purine ring is constructed by a series of reactions that add the donated carbons and nitrogens to a preformed ribose 5- phosphate. •N atoms from aspartic acid, glycine, and glutamine),
10 • C atoms from CO2, and N – formyltetrahydrofolate are added to PRPP to form intermediates until IMP is formed Synthesis of 5′-phosphoribosylamine: This occurs when the amide group of glutamine replaces the pyrophosphate group attached to carbon 1 of PRPP. The reaction is catalysed by PRPP Glutamyl amidotransferase This is the committed step in purine nucleotide biosynthesis The enzyme is inhibited by AMP, GMP, and inosine monophosphate (IMP) Synthesis of 5′-phosphoribosylamine contd… Synthesis of 5′-phosphoribosylamine contd… The rate of the reaction is also controlled by the intracellular concentration of PRPP. The intracellular concentration of PRPP is normally far
below the Km for the amidotransferase. Any small change in the PRPP concentration causes a proportional change in the rate of the reaction Synthesis of Inosine Monophosphate: The synthesis of inosine monophosphate from 5′- phosphoribosylamine requires a series of nine steps Inosine monophosphate is considered as a “parent” purine nucleotide Five ATP molecules are used as an energy source There are two steps in the pathway that require N10- formyltetrahydrofolate i.e. i. The conversion of glycinamide ribosyl-5-phosphate to formylglycinamide ribosyl-5-phosphate ii. The conversion of aminoimidazole carboxamide ribosyl-5- phosphate to formimidoimidazole carboxamide ribosyl-5- phosphate Both reactions are catalysed by the enzyme formyl transferase
The activities that catalyze reactions 2, 3, and 5 are all contained in a single tri-functional enzyme encoded by the GART gene 2, phosphoribosylglycinamide formyltransferase, (5′- phosphoribosylamine to Glycinamide ribonucleotide) 3, phosphoribosylglycinamide synthetase, (Glycinamide ribonucleotide to Formylglycinamide ribonucleotide) 4, phosphoribosylformylglycinamide synthase (Formylglycinamide ribonucleotide to Formylglycinamidine ribonucleotide) 5, phosphoribosylaminoimidazole synthetase (Formylglycinamidine ribonucleotide to 5 aminoimidazole ribonucleotide)
6, phosphoribosylaminoimidazole carboxylase, (5 aminoimidazole ribonucleotide to Carboxyaminoimidazole ribonucleotide)
7, phosphoribosylaminoimidazole succinocarboxamide synthetase (Carboxyaminoimidazole ribonucleotide to N-succinyl -5-aminoimidazole-4-carboxamide ribonucleotide) 6&7 bi-functional enzyme encoded by the PAICS gene
8, Adenylosuccinate lyase (N-succinyl -5- aminoimidazole-4-carboxamide ribonucleotide to 5-aminoimidazole-4-carboxamide ribonucleotide)
9, 5-aminoimidazole-4-carboxyamide ribonucleotide formyltransferase (5-aminoimidazole-4-carboxamide ribonucleotide to N formyl aminoimidazole-4- carboxamide ribonucleotide
10, IMP cyclohydrolase / IMP synthase (N formyl aminoimidazole-4-carboxamide ribonucleotide to IMP) NB: 9 and 10 ------a bi-functional enzyme encoded by the ATIC gene IMP represents a branch point for purine biosynthesis Conversion of IMP to AMP and GMP: The conversion of IMP to either AMP or GMP uses a two- step, energy-requiring pathway. The synthesis of AMP requires guanosine triphosphate (GTP) as an energy source The synthesis of GMP requires ATP as an energy source The first reaction in each pathway is inhibited by the end product of that pathway. This gives the cell a mechanism for diverting IMP to the synthesis of the species of purine present in lesser amounts. If both AMP and GMP are present in adequate amounts, the de novo pathway of purine synthesis is turned off at the amidotransferase step.
Formation of nucleoside diphosphates and triphosphates: Nucleoside diphosphates (NDP) are synthesized from the corresponding nucleoside monophosphates (NMP) by base-specific nucleoside monophosphate kinases These kinases do not discriminate between ribose or deoxyribose in the substrate ATP serves as the source of the transferred phosphate This occurs because ATP is present in higher concentrations than the other nucleoside triphosphates. Adenylate kinase is particularly active in liver and muscle, where the turnover of energy from ATP is high.
Formation of nucleoside diphosphates and triphosphates: The function of Adenylate kinase is to maintain an equilibrium among AMP, ADP, and ATP.
Guanylate Kinase,
Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase
Nucleoside diphosphate kinase, unlike the monophosphate kinases, has broad specificity. Catabolism of purine bases/nucleotides: Purines that result from the normal turnover of cellular nucleic acids, or that are obtained from the diet and not degraded, can be converted to nucleoside triphosphates and used by the body.
The degradation of the purine nucleotides (AMP and GMP) occurs mainly in the liver
This involves conversion of purines, their ribonucleosides, and their deoxyribonucleosides to mononucleotides.
The process requires far less energy than the de novo pathway
The purine mononucleotides, AMP, GMP, IMP, and XMP are all catabolized to uric acid
Catabolism of purine bases/nucleotides Each mononucleotide is first converted to the phosphate free nucleoside form through the actions of one of several cytosolic 5'-nucleotidases.
The nitrogen is removed from adenosine generating inosine by enzyme adenosine deaminase (AMP deaminase) ADA.
Loss of ADA activity results in the potentialy lethal disorder, Severe Combined Immunodeficiency disease (SCID) OR AMP is first deaminated to produce IMP (AMP deaminase)
Then IMP and GMP are dephosphorylated (5-nucleotidase)
Catabolism of purine bases/nucleotides The ribose is cleaved from the nucleosides (IMP & GMP) by purine nucleoside phosphorylase (PNP) to yield Hypoxanthine and Guanine.
Hypoxanthine, produced by cleavage of IMP, is converted to xanthine by xanthine oxidase
The nitrogen is removed from guanine by guanine deaminase (enzyme guanase) yielding xanthine.
The pathways for the degradation of adenine and guanine merge at this point
Catabolism of purine bases contd… Hypoxanthine and xanthine are then converted to the terminal product of purine catabolism, uric acid, by the enzyme xanthine oxidase.
Little energy is derived from the degradation of the purine ring Thus, it is to the cell’s advantage to recycle and salvage the ring Salvage of purine nucleotides One important mechanism involves
phosphoribosylation of a free purine by PRPP
And 2 (Enzymes) classified as phosphoribosyl transferases are involved in phosphoribosylation
1st catalyses the conversion of adenine to AMP.
2nd catalyses the conversion hypoxanthine and guanine to IMP or GMP respectively.
Salvage of purine nucleotides contd…. Two enzymes are involved, namely; i. Adenine phosphoribosyltransferase ii. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Both enzymes use PRPP as the source of the ribose 5-phosphate group. The release of pyrophosphate and its subsequent hydrolysis by pyrophosphatase makes these reactions irreversible
Salvage of purine nucleotides contd…. A second salvage mechanism involves phosphoryl transfer from ATP to a purine ribonucleoside
Adenosine kinase catalyzes phosphorylation of adenosine and deoxyadenosine to AMP and dAMP
Deoxycytidine kinase phosphorylates deoxycytidine and 2′-deoxyguanosine to dCMP and dGMP. Role of Ribonucleotide reductase enzyme in salvage pathway:
This enzyme is specific for the reduction of nucleoside diphosphates (adenosine diphosphate (ADP) and guanosine diphosphate (GDP), to their deoxy-forms (dADP, dGDP,) The immediate donors of the hydrogen atoms needed for the reduction of the 2′-hydroxyl group are two sulfhydryl groups on the enzyme itself During the reaction, the sulfhydryl groups form a disulfide bond
Regeneration of reduced enzyme:
In order for ribonucleotide reductase to continue producing deoxyribonucleotides, the disulfide bond created during the production of the 2′-deoxy carbon must be reduced. The source of the reducing equivalents for this purpose is thioredoxin—a peptide coenzyme of ribonucleotide reductase. Thioredoxin contains two cysteine residues separated by two amino acids in the peptide chain. The two sulfhydryl groups of thioredoxin donate their hydrogen atoms to ribonucleotide reductase, in the process forming a disulfide bond Dependance on salvage pathway: The liver provides purines and purine nucleosides for salvage and utilization by tissues incapable of their biosynthesis. The human brain has a low level of PRPP amidotransferase and hence depends in part on exogenous purines. Erythrocytes and polymorphonuclear leukocytes cannot synthesize 5-phosphoribosylamine These therefore utilize exogenous purines to form nucleotides. Regulation of Purine Synthesis: Regulation of purine synthesis occurs at several sites Four key enzymes are regulated: i. PRPP synthetase ii. Amidophosphoribosyl transferase iii. Adenylosuccinate synthetase iv. IMP dehydrogenase The first two enzymes regulate IMP synthesis The last two regulate the production of AMP and GMP, respectively.
Synthetic inhibitors of purine synthesis: Sulfonamides: They are structural analogs of para-aminobenzoic acid. (PABA) They competitively inhibit bacterial synthesis of folic acid. Sulfonamides are C-Ihs with enzyme Dihydropteroate synthase DHPS Since purine biosynthesis requires tetrahydrofolate as a coenzyme, the sulfa drugs slow down this pathway in bacteria Sulfa drugs cannot interfere with human purine biosynthesis reactions because humans depend on external sources of folic acid
Synthetic inhibitors of purine synthesis contd…. Methotrexate: This compound is a structural analog of folic acid It is used pharmacologically to control the spread of cancer by interfering with the synthesis of nucleotides By doing so, it therefore interfere with synthesis of DNA and RNA Trimethoprim: It is another folate analog Has potent antibacterial activity because of its selective inhibition of bacterial dihydrofolate reductase. A major disadvantage of the folic acid analogues is toxicity to all dividing cells They Inhibits Dihydrofolate reductase so no active THF (5,6,7,8Tetrahydrofolic acid ) formed
DISORDERS OF PURINE METABOLISM: Genetic diseases of purine metabolism include: i. Gout ii. Lesch-Nyhan syndrome iii. Adenosine deaminase deficiency iv. Purine nucleoside phosphorylase deficiency Gout: •Gout is a disease of the joints caused by an elevated concentration of uric acid in the blood and tissues
•When serum urate levels exceed the solubility limit, sodium urate crystalizes in soft tissues and joints
•The joints become inflamed, painful, and arthritic, owing to the abnormal deposition of sodium urate crystals.
•However, most cases of gout reflect abnormalities in renal handling of uric acid •Gout occurs predominantly in males
•Most forms of gout are the result of excess purine production and consequent catabolism or to a partial deficiency in the salvage enzyme, HGPRT Gout contd…. •The kidneys are also affected, as excess uric acid is deposited in the kidney tubules
•In most mammals and many other vertebrates, uric acid is further degraded to allantoin by the action of urate oxidase
•Gout is effectively treated by a combination of nutritional and drug therapies
•Foods especially rich in nucleotides and nucleic acids, such as liver or glandular products, are withheld from the diet
•Major alleviation of the symptoms is provided by the drug allopurinol
Gout contd…. The drug inhibits xanthine oxidase, the enzyme that catalyzes the conversion of purines to uric acid
Allopurinol is a substrate of xanthine oxidase, which converts allopurinol to oxypurinol (alloxanthine)
Oxypurinol inactivates the reduced form of the enzyme by remaining tightly bound in its active site.
When xanthine oxidase is inhibited, the excreted products of purine metabolism are xanthine and hypoxanthine These are more water soluble than uric acid and less likely to form crystalline deposits Lesch-Nyhan Syndrome: This condition reflects a defect in hypoxanthine- guanine phosphoribosyl transferase(HGPRT), an enzyme of purine salvage pathway It is seen almost exclusively in male children It is a hyperuricemia characterized by frequent episodes of uric acid lithiasis and a bizarre syndrome of self-mutilation There is an accompanying rise in intracellular PRPP, which results in purine overproduction Lesch-Nyhan Syndrome: In addition, there is decreased IMP and GMP levels As a result PRPP Glutamyl amidotransferase(the committed step in purine synthesis) has excess substrate and decreased inhibitors available This results in increased de novo purine synthesis The combination of decreased purine reutilization and increased purine synthesis results in increased degradation of purines and the production of large amounts of uric acid Children with this genetic disorder, which becomes manifest by the age of 2 years, are sometimes poorly coordinated and mentally retarded
Lesch-Nyhan Syndrome contd.. •They are extremely hostile and show compulsive self-destructive tendencies
•They mutilate themselves by biting off their fingers, toes, and lips
•The devastating effects of Lesch- Nyhan syndrome illustrate the importance of the salvage pathways
•The brain is especially dependent on the salvage pathways, and this may account for the central nervous system damage in children with Lesch-Nyhan syndrome Lesch-Nyhan Syndrome contd..
In patients with Lesch-Nyhan syndrome, the hyperuricemia frequently results in the formation of uric acid stones in the kidneys There is also deposition of urate crystals in the joints (gouty arthritis) and soft tissues In addition, the syndrome is characterized by motor dysfunction, cognitive deficits and behavioral disturbances Hyporuricemia: Hypouricemia and increased excretion of hypoxanthine and xanthine are associated with xanthine oxidase deficiency It can be due to a genetic defect or to severe liver damage Patients with a severe enzyme deficiency may exhibit xanthinuria and xanthine lithiasis Adenosine Deaminase Deficiency:
•A deficiency in adenosine deaminase activity leads to severe combined immunodeficiency disease (SCID)
•In this immunodeficiency, both thymus derived lymphocytes (T cells) and bone marrow-derived lymphocytes (B cells) are sparse and dysfunctional.
•This leaves the individual without a functional immune system
•Children born with this disorder lack a thymus gland Adenosine Deaminase Deficiency:
They become subject to many opportunistic infections because of the lack of a functional immune system
Death results if the child is not placed in a sterile environment
The disease is treated by a adminstering a modified form of adenosine deaminase enzyme
Lack of Adenosine deaminase enzyme leads to a 100-fold increase in the cellular concentration of dATP, a strong inhibitor of ribonucleotide reductase
Nucleoside Phosphorylase Deficiency (PNP)
This condition is associated with a severe deficiency of T cells but apparently normal B cell function Immune dysfunctions appear to result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase and thereby deplete cells of DNA precursors. Disorder Defect Nature of Defect Comments
Gout PRPP synthetase increased enzyme activity hyperuricemia
Gout HGPRT Partial enzyme deficiency hyperuricemia
Gout glucose-6-phosphatase enzyme deficiency hyperuricemia
Lesch-Nyhan HGPRT lack of enzyme hyperuricemia syndrome SCID ADA lack of enzyme
Immunodeficiency PNP lack of enzyme
2,8-dihydroxyadenine, Renal lithiasis APRT lack of enzyme renal lithiasis hypouricemia and xanthine Xanthinuria Xanthine oxidase lack of enzyme renal lithiasis von Gierke disease Glucose-6-phosphatase enzyme deficiency Synthesis of Pyrimidine nucleotides: De novo pathways: In the synthesis of the pyrimidine nucleotides, the base is synthesized first, and then it is attached to the ribose 5-phosphate moiety The ribose 5-phosphate moiety is donated by PRPP The sources of the atoms in the pyrimidine ring are
glutamine, CO2, and aspartic acid Glutamine and aspartic acid are thus required for both purine and pyrimidine synthesis Sources of the atoms for pyrimidine ring: Reactions of the pathway: In the initial reaction of the pathway, glutamine combines with bicarbonate (CO2) and ATP to form carbamoyl phosphate. This reaction is analogous to the first reaction of the urea cycle, except that it uses glutamine as the source of the nitrogen (rather than ammonia) It occurs in the cytosol (rather than in mitochondria). The reaction is catalyzed by carbamoyl phosphate synthetase II, which is the regulated step of the pathway. The analogous reaction in urea synthesis is catalyzed by carbamoyl phosphate synthetase I
De novo pathways contd…. In the next step of pyrimidine biosynthesis, the entire aspartate molecule adds to carbamoyl phosphate in a reaction catalyzed by aspartate transcarbamoylase. The molecule subsequently closes to produce a ring (catalyzed by dihydroorotase) The ring is then oxidized to form orotic acid (or its anion, orotate) through the actions of dihydroorotate dehydrogenase(found on the inner mitochondrial membrane) The enzyme orotate phosphoribosyl transferase catalyzes the transfer of ribose 5-phosphate from PRPP to orotate, producing orotidine 5-phosphate De novo pathways contd…. Orotidine 5-phosphate is decarboxylated by orotidylic acid decarboxylase to form uridine monophosphate (UMP) In mammals, the first three enzymes of the pathway (carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorotase) are located on the same polypeptide, designated as CAD. The last two enzymes of the pathway are similarly located on a polypeptide known as UMP synthase De novo pathways contd…. UMP is phosphorylated to UTP. An amino group, derived from the amide of glutamine, is added to carbon 4 to produce CTP by the enzyme CTP synthetase UTP and CTP are precursors for the synthesis of RNA
Synthesis of thymidine monophosphate (TMP) from dUMP: dUMP is converted to dTMP by thymidylate synthase
Methylenetetrahydrofolate transfers a methyl group to dUMP to form dTMP
The enzyme uses N5,N10-methylene tetrahydrofolate as the source of the methyl group
Phosphorylation reactions then produce dTTP, a precursor for DNA synthesis
dTTP is also a regulator of ribonucleotide reductase. Regulation of De Novo Pyrimidine Synthesis:
The regulated step of pyrimidine synthesis in humans is carbamoyl phosphate synthetase II The enzyme is inhibited by UTP and activated by PRPP As pyrimidines decrease in concentration (as indicated by UTP levels), CPS II is activated and pyrimidines are synthesized The activity is also regulated by the cell cycle Regulation of De Novo Pyrimidine Synthesis contd…. As cells approach S-phase, CPS-II becomes more sensitive to PRPP activation and less sensitive to UTP inhibition At the end of S-phase, the inhibition by UTP is more pronounced, and the activation by PRPP is reduced. Salvage of Pyrimidine Bases: Few pyrimidine bases are salvaged in human cells Pyrimidine bases are normally salvaged by a two-step route. First, a relatively nonspecific pyrimidine nucleoside phosphorylase converts the pyrimidine bases to their respective nucleosides The more specific nucleoside kinases then react with the nucleosides, forming nucleotides This route for synthesis of pyrimidine nucleoside monophosphates is relatively inefficient for salvage of pyrimidine bases Salvage of Pyrimidine Bases contd. This is because of the very low concentration of the bases in plasma and tissues Pyrimidine phosphorylase can use all of the pyrimidines but has a preference for uracil It is sometimes called uridine phosphorylase The phosphorylase uses cytosine fairly well but has a very, very low affinity for thymine Salvage of Pyrimidine Bases contd. Special features of thymidine kinase: This enzyme is allosterically inhibited by dTTP. Activity of thymidine kinase in a given cell is closely related to the proliferative state of that cell. During the cell cycle, the activity of TK rises dramatically As cells enter S phase, the levels of this enzyme increase Generally, rapidly dividing cells have high levels of this enzyme CATABOLISM OF PYRIMIDINE BASES: The pyrimidine nucleotides are dephosphorylated, and the nucleosides are cleaved to produce ribose 1- phosphate and the free pyrimidine bases cytosine, uracil, and thymine Cytosine is deaminated, forming uracil, which is converted to CO2, NH4+, and β-alanine Thymine is converted to CO2, NH4+, and β- aminoisobutyrate These products of pyrimidine degradation are excreted in the urine or converted to CO2, H2O, and NH4+(which forms urea). Catabolism of pyrimidine bases contd… Catabolism of pyrimidine bases contd… They do not cause any problems for the body, in contrast to uric acid, which is produced from the purines Uric acid can precipitate as sodium urate crystals, which causes gout As with the purine degradation pathway, little energy can be generated by pyrimidine degradation Orotic aciduria: It is a rare genetic defect caused by deficiency of the bifunctional enzyme called UMP Synthase. The UMP Synthase enzyme has two catalytic domains namely; i. Orotate phosphoribosyltransferase ii. Orotidylate decarboxylase In this condition, large amounts of orotic acid are excreted in the urine The disease is characterized by poor growth (because pyrimidines cannot be synthesized) and megaloblastic anaemia Alternative cause of orotic aciduria: When ornithine transcarbamoylase is deficient (urea cycle disorder), excess carbamoyl phosphate from the mitochondria leaks into the cytoplasm The elevated levels of cytoplasmic carbamoyl phosphate lead to pyrimidine production This pyrimidine production occurs as the regulated step of the pathway, the reaction catalyzed by carbamoyl phosphate synthetase II, is being bypassed Thus, orotic aciduria results Catalytic activity of UMP Synthase: Management of orotic aciduria: Oral adminstration of uridine has been shown to result in improvement of the anaemia It also causes decreased excretion of orotic acid in the urine Uridine, which is converted to UMP, bypasses the metabolic block In doing this, it provides the body with a source of pyrimidines, as both CTP and dTMP can be produced from UMP.