Nucleotide Metabolism -Biosynthesis- Dr. Sooad Al-Daihan 1

Metabolic Pathways The biochemical reactions in the living cell are organized into metabolic pathways. The pathways have dedicated purposes: Extraction of energy. Storage of fuels. Synthesis of important building blocks. Elimination of waste materials. The pathways can be represented as a map: Follow the fate of metabolites and building blocks. Identify enzymes that act on these metabolites. Identify points and agents of regulation. Identify sources of metabolic diseases. Lecture no.7

Homeostasis Organisms maintain homeostasis by keeping the concentrations of most metabolites at steady state. In steady state, the rate of synthesis of a metabolite equals the rate of breakdown of this metabolite. Pathways are at steady state unless perturbed. After perturbation a NEW steady state will be established. The flow of metabolites through the pathways is regulated to maintain homeostasis. Sometimes, the levels of required metabolites must be altered very rapidly. Lecture no.7

Feedback Inhibition In many cases, ultimate products of metabolic pathways directly or indirectly inhibit their own biosynthetic pathways. Lecture no.7

Rate of reaction depends on the concentration of substrates The rate is more sensitive at low substrate concentrations. Frequency of substrate meeting the enzyme matters. The rate becomes insensitive at high substrate concentrations. The enzyme is nearly saturated with substrate. Lecture no.7

Significances of nucleotides They are precursors of DNA and RNA They are the energy currency in metabolic transactions. They are components of: – Cofactors : such as NAD, FAD, S-adenosylmethionine, and coenzyme A – Activated biosynthetic intermediates : such as UDP-glucose and CDP-diacylglycerol. – Second messengers : such as cAMP and cGMP 6 Lecture no.7

Overview of Nucleotide Metabolism 7 Lecture no.7

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Nucleotide Biosynthesis 9 Begins with their metabolic precursors: amino acids, ribose 5- phosphate, CO 2, and NH 3. Recycles the free bases and nucleosides released from nucleic acid breakdown Question: Why do we need both pathways? Lecture no.7

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In these pathway, two features deserve to mention: First, there is evidence, especially in the de novo purine pathway, that the enzymes are present as large, multienzyme complexes in the cell. Second, the cellular pools of nucleotides (other than ATP) are quite small, 1% or less of the amounts required to synthesize the cell’s DNA. Therefore, cells must continue to synthesize nucleotides, and in some cases nucleotide synthesis may limit the rates of DNA replication and transcription. 11 Because of the importance of these processes in dividing cells, agents that inhibit nucleotide synthesis have become particularly important to modern medicine. Lecture no.7

Denovo Purine Nucleotide Biosynthesis 12 The two parent purine nucleotides of nucleic acids are adenosine 5-monophosphate (AMP) and guanosine 5- monophosphate (GMP). The origin of the carbon and nitrogen atoms of the purine ring system, as determined by John Buchanan using isotopic tracer experiments in birds

Continue… Synthesis of Inosine Monophosphate (IMP) Basic pathway for biosynthesis of purine ribonucleotides. Starts from ribose-5-phosphate which is derived from the PPP. Requires 11 steps overall. Occurs primarily in the liver. Lecture no.7

Step 1: Activation of ribose-5-phosphate Enzyme: Ribose phosphate pyrophosphokinase Product: 5-phosphoribosyl-a-pyrophosphate (PRPP). PRPP is also a precursor in the biosynthesis of pyrimidine nucleotides and the amino acids histidine & tryptophan. This step is tightly regulated by feedback inhibition. Lecture no.7

Step 2: Acquisition of purine atom 9 In this committed step, an amino group donated by glutamine is attached at C-1 of PRPP. Enzyme: Amidophosphoribosyl transferase. Product: Resulting in the formation of 5-phosphoribosylamine. What is the properties of committed step Lecture no.7

Step 3: Acquisition of purine atoms C4, C5, and N7 Phosphoribosylamine reacts with ATP and glycine to produce glycinamide ribonucleotide (GAR). Enzyme: Glycinamide synthetase. Lecture no.7

Step 4: Acquisition of purine atom C8 Formylation of free α-amino group of GAR. Enzyme: GAR transformylase. Co-factor of enzyme : N 10 -formyl THF Step 5: Acquisition of purine atom N3 The amide amino group of a second glutamine is transferred to form formylglycinamidine ribonucleotide (FGAM). Enzyme: FGAM synthetase. Step 6: Closing the ring Closing of the imidazole ring or formation of 5-aminoimidazole ribonucleotide (AIR). Enzyme: AIR synthetase. Lecture no.7

Step 7: Acquisition of C6 C6 is introduced as HCO 3 -. The reaction is driven by hydrolysis of ATP. Enzyme: AIR carboxylase (aminoimidazole ribonucleotide carboxylase). Product: CAIR (carboxyaminoimidazole ribonucleotide). Lecture no.7

Step 8: Acquisition of N1 N1 is acquired from aspartate in an amide condensation reaction that is driven by hydrolysis of ATP to produce 5-aminoimidazole-4-(N- succinylocarboxamide) ribonucleotide. Enzyme: SAICAR synthetase Step 9: Elimination of fumarate by the action of adenylosuccinate lyase to produce 5- aminoimidazole-4-carboxamide ribonucleotide (AICAR) Step 10: Acquisition of C2 Another formylation reaction catalyzed by AICAR transformylase results in the formation of 5-formylaminoimidazole- 4-carboxamide ribonucleotide (FAICAR)

Biochemistry in medicine 20 The dependence of purine biosynthesis on folic acid compounds at Steps 4 and 10 means that antagonists of folic acid metabolism indirectly inhibit purine formation and, in turn, nucleic acid synthesis, cell growth, and cell division. Clearly, rapidly dividing cells such as malignancies or infective bacteria are more susceptible to these antagonists than slower- growing normal cells. Lecture no.7

Step 11: Cyclization or ring closure Water is eliminated by the action of inosine monophosphate (IMP) synthase. In contrast to step 6 (closure of the imidazole ring), this reaction does not require ATP hydrolysis. Once IMP is formed, it is rapidly converted to AMP and GMP. Lecture no.7

Biosynthesis of AMP and GMP from IMP Lecture no.7

Purine nucleotide biosynthesis is regulated by feedback control The significance of regulation: (1) Fulfill the need of the body, without wasting. (2) [GTP]=[ATP] Lecture no.7

Salvage Pathway of Purine Two phosphoribosyl transferases are involved in this pathway: Adenosine phosphoribosyl transferase (APRT) Adenine + PRPP AMP + Ppi Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Hypoxanthine + PRPP IMP + Ppi Guanine + PRPP GMP + Ppi APRT is not very important because it generate little adenine HGPRT, is exceptionally important and it is inhibited by both IMP and GMP Lecture no.7

Mutations in genes that encode nucleotide biosynthetic enzymes can reduce levels of needed nucleotides and can lead to an accumulation of intermediates. Lesch-Nyhan syndrome is compulsive self-destructive behavior, caused by a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of salvage pathway, The disease is inherited as a sex-linked recessive disorder. In the absence of HGPRT, PRPP levels rise and purines are overproduced by the de novo pathway, resulting in high levels of uric acid production and gout-like damage to tissue. 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. Continue… Continue… Lecture no.7

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Continue… 27 Lecture no.7

Pyrimidine Denovo Synthesis It is a shorter pathway than for purines. The base is made first, then attached to ribose-P (unlike purine biosynthesis). Requires 6 steps (instead of 11 for purine). The product is UMP (uridine monophosphate). Only 2 precursors (aspartate and glutamine, plus HCO 3 - ) contribute to the 6-membered ring. Lecture no.7

Pyrimidine Denovo Synthesis Lecture no.7

Nucleotide Mono-, Di-, and Triphosphates Are Interconvertible How is the other major pyrimidine ribonucleotide, cytidine, formed? It is synthesized from the uracil base of UMP, but UMP is converted into UTP before the synthesis can take place. The di and triphosphates are the active forms of nucleotides in biosynthesis and energy conversions. Lecture no.7

Continue… 31 Nucleoside monophosphates are converted into nucleoside triphosphates in stages. First, nucleoside monophosphates are converted into diphosphates by specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl-group donor. For example: UMP + ATP UDP + ADP Second, Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase, an enzyme that has broad specificity, in contrast with the monophosphate kinases. XDP + YTP XTP + YDP UMP kinase nucleoside diphosphate kinase Lecture no.7

32 CTP Is Formed by Amination of UTP After uridine triphosphate has been formed, it can be transformed into cytidine triphosphate by the replacement of a carbonyl group by an amino group. This reaction requires ATP and uses glutamine as the source of the amino group. CTP can be used then in many biochemical processes, including RNA synthesis. Lecture no.7

Thymidylate Is Formed by the Methylation of Deoxyuridylate 33 Uracil, produced by the pyrimidine synthesis pathway, is not a component of DNA. Rather, DNA contains thymine, a methylated analog of uracil. Another step is required to generate thymidylate from uracil. Thymidylate synthase catalyzes this step -deoxyuridylate (dUMP) is methylated to thymidylate (TMP). The methylation of this nucleotide facilitates the identification of DNA damage for repair and, hence, helps preserve the integrity of the genetic information stored in DNA. Lecture no.7

34 Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase. In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from the N 5,N 10 -methylenetetrahydrofolate, as shown in red and gray. Continue…

Pyrimidine Salvage pathway 35 Lecture no.7

Nucleotide Metabolism -Degradation- Lecture no.7

Purine Catabolism Purine nucleotides (AMP and GMP)are degraded by a pathway in which they lose their phosphate through the action of 5’- nucleotidase,to form adenosine and guanosine respectively. Adenosine  It is deaminated to inosine by adenosine deaminase.  Inosine is hydrolyzed to hypoxanthine and D-ribose by the action of nucleosidase.  Hypoxanthine is oxidized successively to xanthine and then uric acid by xanthine oxidase Guanosine  It is cleaved to guanine and D-ribose by the action of nucleosidase.  Guanine undergoes hydrolytic removal of its amino group to yield xanthine by the action of guanine deaminase.  Xanthine is converted to uric acid by xanthine oxidase. Lecture no.7

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Purine Metabolism Disorders Genetic aberrations in human purine metabolism have been found, some with serious consequences. For example, adenosine deaminase (ADA) deficiency.  The absence of ADA leads to severe combined immunodeficiency disease (SCID) in which T lymphocytes and B lymphocytes do not develop properly.  Lack of ADA leads to a 100-fold increase in the cellular concentration of dATP, a strong inhibitor of ribonucleotide reductase involved in deoxynucleotide biosynthesis  Infants with this deficiency have a high fatality rate due to infections.  It is treated by administering ADA which can remain in the blood for 1 – 2 weeks, or by gene therapy where the gene that is missing or defective is replaced Lecture no.7

GOUT Is a disease of the joints caused by an elevated concentrations of uric acid in the blood and tissues. The joints become inflamed, painful, and arthritic, owing to the abnormal disposition of crystals of monosodium urate monohydrate. Usually affect joints in the lower extremities (the big toe is the classic site). Lecture no.7

Allopurinol It is a structural analog of hypoxanthine that strongly inhibit xanthine oxidase. It is used to prevent of attacks of gouty arthitis and nephropathy It is also used during chemotherapy of cancer and to prevent recurrent calcium oxalate calculi. Lecture no.7

Allopurinol Pathway Lecture no.7

Catabolism of a 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. Lecture no.7

Ring Cleavage In order for the rings (Cytosine and Thymine) to be cleaved, they must first be reduced by NADPH. Atoms 2 and 3 of both rings are released as ammonia and carbon dioxide. The rest of the ring is left as a beta-amino acid. Beta-amino isobutyrate from thymine or 5-methyl cytosine is largely excreted. Beta-alanine from cytosine or uracil may either be excreted or incorporated into the brain and muscle dipeptides, carnosine (his-beta-ala) or anserine (methyl his-beta-ala). Lecture no.7