PHBC 522 Biochemistry I Lecture 7 - Nucleotide Metabolism (Winter 2024) PDF

PHBC 522 – Biochemistry I PHBC 522 – Winter 2024 Faculty for Pharmacy and Biotechnology Lecture 7 – Nucleotide Metabolism Prof. Sahar Mohamed 1  Learning outcomes By the end of the lecture you should be able t...

PHBC 522 – Biochemistry I PHBC 522 – Winter 2024 Faculty for Pharmacy and Biotechnology Lecture 7 – Nucleotide Metabolism Prof. Sahar Mohamed 1  Learning outcomes By the end of the lecture you should be able to: Draw the structure of purines and pyrimidines. Mention the different biosynthesis pathways of purines and pyrimidines. Describe the regulations and feedback inhibition in the biosynthesis of nucleotides. Know Clinical disorders of purine degradation. Describe the clinical manifestations of leshnyhan syndorme and gout. Know how to treat hyperuriceamia and gout. Show how nucleotides are digested in the small intestine. 2  Importance of nucleotides Nucleotides are the building units of DNA and RNA. They are necessary for the replication of the genome & the transcription of genetic information. They serve as carriers of activated intermediates in the synthesis of some carbohydrates, lipids and proteins as UDP-glucose which participated in the formation of glycogen. Also, they are structural components of several essential coenzymes, for example Coenzyme A, FAD, NAD+ and NADH+. Nucleotides form ATP which is the universal currency of energy & GTP which is also an energy source. ATP also acts as a phosphoryl donor transferred by protein kinase. Nucleotides as cAMP and cGMP, serve as 2nd messenger in signal transduction pathways. Nucleotide biosynthesis pathways are tremendously important because many of the most widely used drugs in the treatment of cancer blocks steps in its biosynthesis, particularly steps in the synthesis of DNA. 3  Nucleotide Structure Nucleotide Nitrogenous Base Pentose Sugar Phosphate Purines or pyrimidines ribose or deoxyribose 1Por 2P or 3P 4 5 Unusual Bases 6  N-9 glycosidic linkage N-1 glycosidic linkage Reduced Carbon OH A- Pentoses found in nucleic acids B- Examples of the numbering systems of purine- and pyrimidine-containing nucleosides. 7 5’ CH2 8 9  Biosynthesis of nucleotides – two pathways, one precursor 1-De Novo pathways: synthesis of nucleotides from simpler starting materials: Activated ribose (PRPP) + amino acids + ATP + CO 2 + …  Nucleotides De Novo synthesis requires energy (ATP), numerous precursors and enzymes. 2-Salvage pathways: synthesis of nucleotides from already preformed bases after their turnover. Activated ribose (PRPP) + bases  Nucleotides Salvage pathways use already made bases, so require much less energy and enzymes. Up to 90 % of nucleotides may be synthesized through salvage pathways. Phosphoribosyl pyrophosphate (PRPP) (the activated form of ribose) that is required for the biosynthesis of all nucleotides. It is synthesized from ribose-5-phosphate (from pentose phosphate pathway) and ATP (note: transfer of pyrophosphate, PPi). Purine Pi ribonucleotides _ + 10  Biosynthesis of pyrimidine nucleotides – step 1 The first step in pyrimidine biosynthesis is synthesis of carbamoyl phosphate. Note that this reaction occurs in the cytosol, catalyzed by CPS II (using glutamine as N- source) for pyrimidine synthesis. (Urea synthesis: CPS I, using NH4+ as N-source, reaction takes place in mitochondria) The synthesis is a variation of a general theme in nucleotide biosynthesis: - activation of Carbonyl-O as phosphate (enol phosphate  phosphoryl transfer potential) - nucleophilic displacement of phosphate (note: no net oxidation/reduction). 11 Carbamoyl phosphate synthetase – (3 active sites) 12  Biosynthesis of pyrimidine nucleotides – step 2 In the second step of pyrimidine biosynthesis, carbamoyl phosphate is coupled to aspartate; ring closure and dehydration yield orotate. This reaction is catalyzed by transcarbomylase. Note again the coupling technique: R–OH  R–O-PO3-  R–Nu + PO4- 13 Biosynthesis of pyrimidine nucleotides – step 3 Orotic aciduria Low activities of orotate Phosphoribosyl- Transferase and Orotydilate decarboxylase result in abnormal growth megaloplastic anemia and excretion of large amt of Orotate in urine. Feeding a diet rich in 14 Uridine improves the case Cytidine is formed from uridine by amination Hmmm, do we recognize something here … ? Cytidine is synthesized from uridine, using ATP for phosphorylation & glutamine as N – (nitrogen) donor and CTP synthase enzyme 15 A Rate limiting step in the synthesis Deoxyribonucleotides (DNA): Reduction of ribonucleotides + H–O–H The substrates for RNR are ADP,GDP,CDP&UDP not TDP, why? 16  Synthesis of Thymidine monophosphate (dTMP from dUMP) dUMP is converted to dTMP by thymidylate synthase, which uses N5,N10-methylene tetrahydrofolate (THF) as the source of the methyl gp. Inhibitors of Thymidine synthase (antitumor) 5- Flurouracil: it is a thymine analogue and it is converted to 5-FdUMP. It is an irreversible inhibitor it binds to the enzyme and makes covalent modification to it and thus cannot be reversed. Methotroxate (MTX): It inhibits the reduction of Dihydrofolate (DHF) to Tetrahydrofolate (THF), therefore decreasing the supply of THF. 17 Regulation of Pyrimidine Biosynthesis: ATP + − CTP PHBC 521 18 18 Salvage pathway of Pyrimidines 19 20 Biosynthesis of purine nucleotides … and again … Hypoxanthine Several steps 21 Inosine Monophosphate (IMP)  Biosynthesis of purine nucleotides 4 Adenylo succinase Adenyloscuccinate synthetase IMP GMP dehydrogenase synthetase IMP Generating AMP and GMP. Inosinate is the precursor of AMP and GMP. AMP is formed by the addition of aspartate followed by the release of fumarate. GMP is generated by the addition of water, dehydrogenation by NAD+, and the replacement of the carbonyl oxygen atom by -NH2 derived from the hydrolysis of glutamine. 22  Purine biosynthesis is regulated by feedback inhibition Control of Purine Biosynthesis. Feedback inhibition by the nucleotide monophosphates controls both the overall rate of purine biosynthesis and the balance between AMP and GMP production. Interplay in the control of nucleotide biosynthesis. Multiple feedback regulation controls synthesis of purine and pyrimidine nucleotides and their reduction to deoxyribonucleotides. Thus a balance in the production of the four nucleotides required for DNA synthesis is maintained. 23  Salvage pathway for synthesis of purine nucleotides 90% of purine nucleotides are formed from salvage pathway. Free purine bases, derived from the turnover of nucleotides or from the diet, can be attached to PRPP to form purine nucleoside monophosphates, in a reaction analogous to the formation of orotidylate. Two salvage enzymes with different specificities recover purine bases. Adenine phosphoribosyltransferase catalyzes the formation of adenylate. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) catalyzes the formation of guanylate as well as inosinate (inosine monophosphate, IMP). 24  Degradation of nucleotides Pyrimidine Catabolism. The pyrimidine ring is completely broken down into CO 2 and NH4+ for degradation/excretion (aspartate  amino acid degradation). 25 Diseases associated with purine degradation HGPRT Guanine Hypoxanthine Purine Degradation 26 Diseases associated with purine degradation ADA Deficiency 27 Diseases associated with purine degradation 28 Degradation of dietary nucleic acids in the small intestine 29  Urate – benefits and diseases In human beings, urate is the final product of purine degradation and is excreted in the urine. High serum levels of urate induce gout, a disease in which salts of urate crystallize and damage joints and kidneys. Allopurinol, an inhibitor of xanthine oxidase, is used to treat gout in some cases. Inhibition of xanthine oxidase leads to reduced levels of uric acids, and higher amounts of hypoxanthine which is better soluble and thus easier excreted. Urate is a highly effective scavenger of reactive oxygen species. Indeed, urate is about as effective as ascorbate (vitamin C) as an antioxidant. The increased level of urate in humans may contribute significantly to the longer life span of humans and to lowering the incidence of human cancer. Mutations in genes that encode nucleotide biosynthetic enzymes can lead to an accumulation of intermediates. A nearly total absence of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) underlies the Lesch-Nyhan syndrome. It is characterised by compulsive self- destructive behavior. At age 2 or 3, children with this disease begin to bite their fingers and lips and will chew them off if unrestrained. These children also behave aggressively toward others. Mental deficiency and spasticity are other characteristics of the disease. Elevated levels of urate in the serum lead to the formation of kidney stones early in life, followed by the symptoms of gout years later. The disease is inherited as a sex-linked recessive disorder. Life expectancy is only ~ 20 years. 30  Summary There are two principal pathways for nucleotide biosynthesis: De novo biosynthesis assembles nucleotides from phosphoribosyl pyrophosphate (activated ribose, PRPP), amino acids, and various donors of 1 to 4 carbon units. Salvage pathways utilise preformed bases (from nucleotide degradation or food) and couple the directly to PRPP. This much more energy-saving patway can contribute up to 90 % of nucleotide synthesis. Pyrimidine nucleotides are synthesised by separate construction of the pyrimidine ring which is then coupled to PRPP. Purine nucelotides are synthesised directly on the sugar template, phosphoribosyl amine, which is derived from PRPP. Deoxyribonucleotides are synthesised from ribonucleotides by reduction. Nucleotide biosynthesis is regulated by product feedback inhibition. The pyrimidine ring is completely broken down into CO2 and NH4+ for degradation. Degradation of purine bases gives urate, which can be excreted without breaking the purine ring system. In humans, urate levels are high (thus gout, the deposition of urate crytals in joints is a frequent disease). Urate, however, is a powerful antioxidant (~ vitamin C). This may be one reason for the increased lifespan of humans. Defects in urate clearance can result in severe disorders: gout, caused by precipitation of urate crystals, and Lesch-Nyhan syndrome, where an inactive salvage pathway enzyme results in multiple defects, including mental deficiency, spasticity, aggressive and self- 31 destructive behaviour, and a reduced life expectancy.  References Lippincott’sIllustrated Reviews:Biochemistry by Richard A. Harvey& Pamela C. Champe. Stryer Biochemistry by L. Stryer, Freeman & Company New York …. Harper’s Biochemistry by R.K. Murray, D.K. Granner, P.A. Mayes & V.W. Rodwell. Biochemistry. Appleton & Lange, New York,Connecticut, California.