NUCLEOTIDE METABOLISM Dr.Samah Badr Msc.MBBS OBJECTIVES: • Introduction • Synthesis of purine nucleotides. • Inhibitors of purine nucleotide synthesis • Regulation of purine nucleotides synthesis • Degradation of purine nucleotides • Disorders of purine metabolism. INTRODUCTION: • Nucleotides: are The building blocks of nucleic acids. • Nucleosides……. • Sugar: can be ribose or deoxy-ribose. • Nitrogen base: two types: NITROGEN BASE: HOW IMPORTANT: • Polymerize to make DNA and RNA • Energy currency of the cell e.g. ATP, GTP • Act as carriers of active intermediates in various metabolic pathways e.g. UDP-glucose in glycogen synthesis, SAM CONT.. • Component of coenzymes e.g. FAD, NADH, NADPH • Act as 2nd messengers e.g. cAMP and cGMP • Allosteric regulation of various metabolic pathways e.g. ATP inhibits PFK-1 DIGESTION: Source: • Nucleotides are both taken in diet and are synthesized in the body. • Dietary nucleotides cannot be used in our body • Human body is exclusively dependent on endogenously synthesized nucleotide for nucleic acid synthesis. CONT… • In food, nucleotides are present mainly in the form of nucleoproteins. • The protein part is degraded by proteolytic enzymes in the GIT. • The nucleic acids are hydrolyzed by nucleases. • DNA is hydrolysed by deoxyribonuclease and RNA by ribonuclease CONT… • Polynucleotidases hydrolyze small poly-nucleotides into mononucleotides. • Nucleotidases hydrolyze nucleotides into nucleosides and phosphate. • Nucleosides are absorbed by mucosal cells of intestine • They are broken down to their final end products in intestinal mucosal cells . • The end products are released into circulation, and are excreted in urine SYNTHESIS OF PURINE NUCLEOTIDES SITE OF SYNTHESIS: ✓ Tissues: • Purines are synthesized by most of the tissues ,the major site is : liver. ✓ Subcellular site: Cytoplasm. ✓ There are two pathways for the synthesis of purines nucleotides: DE NOVO Nucleotides are synthesized afresh from amphibolic inter- SYNTHESIS: mediates (R-5-P). Bases or nucleosides released Salvage from catabolism of pre-existing pathway nucleic acids and nucleotides are reutilized (i.e.recycling) DE NOVO SYNTHESIS • Synthesis of purine nucleotides from various small molecules derived as intermediates of many metabolic pathways in the body. SOURCES OF DIFFERENT ATOMS OF PURINE RING: STEPS OF THE DE NOVO SYNTHESIS: • Starts from the Ribose-5-phosphate that forms 5-phosphoribosyl-1-pyrophosphate (PRPP). • PRPP is required for the synthesis of pyrimidine nucleotides also. • By a series of reactions catalyzed by multifunctional enzymes, PRPP is converted into inosine monophosphate. • Energy consuming process. IMP SIGNIFICANCE: • IMP serves as a precursor for synthesis of all other purine nucleotides such as adenine and guanosine monophosphate (AMP & GMP)and ATP. INHIBITORS OF PURINE SYNTHESIS: Sulfonamides : Methotrexate: • are the structural analogs of Para- • The structural analogs of folic acid aminobenzoic acid (PABA). • Widely used to control cancer • The sulfa drugs can be used to which inhibit the synthesis of inhibit the synthesis of folic acid by purine nucleotides and so nucleic microgranisms that is indirectly acids. reduces the synthesis of purines • These inhibitors also affect the and proliferation of normally growing • sulfonamides have no influence on cells. humans, Why????? • Has many side-effects like: anemia, baldness, scaly skin etc. SALVAGE PATHWAYS FOR PURINE SYNTHESIS • Purine bases created by degradation of RNA and DNA and intermediate of purines synthesis can be directlyconverted to the corresponding nucleotides CONT….. Important pathway: - Save fuel - Some tissues and organs such as brain,RBCs and bone marrow are only capable of synthesizing nucleotides by Salvage Pathways. Purine salvage pathways us one of two enzymes: ✓ Adenine phosphoribosyltransferase (APRT). Converts free adenine to AMP ✓ Hypoxanthine-guanine phosphoribosultransferase (HGPRT). Converts hypoxanthine to IMP and guanine to GMP ThCONT…..e significant of salvage pathway CONT FORMATION OF ATP AND GTP: • AMP and GMP are converted into ADP and GDP respectively by nucleoside monophosphate kinase. • ADP and GDP are converted into ATP and GTP respectively by nucleoside diphosphate kinase ATP & GTP: REGULATION OF PURINE SYNTHESIS: • The synthesis of purine nucleotides is controlled by: – Concentration of PRPP – Feedback regulation at several sites. • Increased concentration of PRPP stimulates the purine nucleotides synthesis. • Concentration of PRPP depends on : – Availability of ribose-5-phosphate – On the activity of PRPP synthase. -Three major feedback mechanisms regulate the overall rate of de novo purine nucleotide synthesis. 1. The step leading to formation of PRPP. This reaction is catalyzed by an allosteric enzyme PRPP synthetase, which is feedback inhibited by purine nucleotides, AMP and GMP (Figure 19.3). 2. The committed step in purine nucleotide biosynthesis is the conversion of PRPP into phosphoribosylamine by PRPP glutamyl-amido transferase which is feedback inhibited by AMP and GMP. 3. AMP and GMP feedback regulate their formation from IMP. AMP feedback regulates adenylosuccinate synthase and GMP feedback regulates IMP dehydrogenase. FORMATION OF DEOXY RIBONUCLEOTIDES: • The synthesis of purine (& pyrimidine )deoxy ribonucleotides occur from ribonucleotides by a reduction at the C2 of ribose moity. • This reaction is catabolised by enzyme ribonucleotide reductase. • The enzyme ribonucleotide reductase itself provides the hydrogen atoms needed for reduction from its sulfhydryl groups. • The reducing equivalents, in turn, are supplied by Thioredoxin, a monomeric protein with two cysteine residues. • NADPH-dependent thioredoxin reductase converts the oxidised thioredoxin to reduced form. DEOXYRIBONUCLEOTIDE SYNTHESIS: DEGREDATION OF PURINE NUCLEOTIDES • The end product of purine metabolism in humans is uric acid. 1. The nucleotide monophosphates (AMP, IMP & GMP ) are converted to their respective nucleoside forms (adenosine,inosine & guanosine ) by the action of nucleosidase. 2. The amino group, either from AMP or adenosine, can be removed to produce IMP or inosine respectively XANTHINE OXIDASE: • Xanthine oxidase is an important enzyme that converts hypoxanthine to xanthine, & xanthine to uric acid. • This enzyme contains FAD, Molybdenum & Iron, & is exclusively found in liver & small intestine. • Xanthine oxidase liberates H2O2 which is harmful to the tissue. • Catlase cleaves H2O2 to H2O & O2. CLINICAL DISORDER RELATED TO PURINE METABOLISM: ✓ HYPERURICEMIA and GOUT ✓ LESCH-NYHAN SYNDROME ✓ KIDNEY STONES ✓ SEVERE COMBINED IMMUNODEFECIENCY (SCID) HYPERURACEMIA: • Characterized by plasma urate (uric acid) level greater than 7.0 mg/dL • Normal plasma levels Females = 2.4 - 6 mg/dL Males = 3.4 - 7 mg/dL GOUT: • Metabolic disease associated with overproduction of uric acid. • Caused by precipitation of sodium urate crystals in the joints resulting in inflammation and pain. • Presenting with painful gouty arthritis affects first metatarsophalangeal joint(GREAT TOE). • This deposit called Tophi. GOUTY TOPHI: TYPES OF GOUT PRIMARY GOUT: It is an inborn error of metabolism due to overproduction of uric acid. Mostly related to over production of purine nucleotides. Secondary gout: Secondary hyperuricemia is due to various diseases causing increased synthesis or decreased excretion of uric acid e.g. cancer like lymphoma ,leukemia… PRIMARY GOUT: • Related to defect in certain enzyme ✓ PRPP synthetase ✓ PRPP glutamylamidotransferse The lack of feedback control of both enzyme by purine nucleotides leads to their elevated synthesis. ✓ HGPRT deficiency: Related to Lesch-Nyhan syndrome MANAGEMENT: ✓ The drug of choice is allopurinol. This is a structural analog of hypoxanthine that competitively inhibits the enzyme xanthine oxidase. Further allopurinol is oxidized to alloxanthine by xanthine oxidase. Alloxanthine, in turn is a more effective inhibitor of xanthine oxidase(suicide inhibition). HypoXanthine and xanthine will accumulated and excreted out. ✓ Low purine diet : Red meat and organ meats (eg. Liver) ,Yeasts and yeast extracts (eg. beer and alcoholic beverages) ,spinach, beans, peas, lentils, oatmeal, cauliflower and mushrooms ✓ Avoid caffeine and alcohol ✓ Keep hydrated LESCH-NYHAN SYNDROME • an a sex-linked metabolic disorder Affect structural gene for HGPRT enzyme located on the X chromosome. • It affects only the males • characterized by excessive uric acid production (often gouty arthritis). Symptoms: - severe gout -severe mental and physical problems - self-mutilating behaviors SEVERE COMBINED IMMUNODEFICIENCY (SCID) • Due to Adenosine deaminase deficiency (ADA)and PNP enzyme. • Accumulation of dATP inhibit ribonucleotide reductase. • Stop DNA replication • Then B and T cells unable to divide. THANK YOU.