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FIFTH WEEK Gihan E-H Gawish, MSc, PhD Ass. Professor Molecular Genetics and Clinical Biochemistry KSU Purine metabolism (Overview) 1. Nomenclature/nucleotide structure 2. Extracellular Hydrolysis of Ingested Nucleic Acid 3. De novo synthesis pathways 4. Re-utilization pathways 5. Metabolic diseases of purine Metabolism (Gout, Lesch-Nyhan, SCID) DNA RNA 1- Endonucleases 2-Phosphodiesterase Oligonucleotides 3- Phosphorylase Ribose + Free Bases Nucleotides Nucleic Acid met abolism Click on any blue rect angle t o see det ails. Carbamoyl Phosphate am i n o a c id s, f o la t e Purine Salvage PRPP Purine Biosynt hesis IMP Pyrimidine Biosynt hesis OMP Pyrimidine Salvage Pyr im idine MP Pur ine MP Ribonucleot ide reduct ase Ribonucleot ide reduct ase dNTP NTP Purine Degradat ion DNA RNA dNTP NTP Pyrimidine Degradat ion NH4 Uric A cid ( energ y ) The nomenclature of purines depends on their linkage to a pentose Adenine Base Adenosine Nucleoside* Base Adenosine Monophosphate Nucleotide Base (P04 ester) * when the base is purine, then the nucleoside ends in OSINE (AdenOSINE, GuanOSINE, InOSINE) The active forms of nucleotides in biosynthesis and energy conversions are di-and tri-phosphates (i) (ii) Nucleoside Monophosphate Kinase CMP + ATP CDP + ADP Nucleoside Diphosphate Kinase XDP + YTP XTP + YDP Structures of Common Purine Bases. H= 6 oxy purine X= 2,6 dioxy purine A= 6 amino purine G= 2 amino, 6-oxy purine Hypoxanthine is an intermediate for Adenine and Guanine (N source) Aspartate (N source) Glutamine The common mechanistic them for the conversion of A and G is the conversion of a carbonyl oxygen to an amino group There are two basic mechanisms to generate purines and pyrimidines 1. DE NOVO BIOSYNTHETIC PATHWAYS (building the bases from simple building blocks) 2. SALVAGE PATHWAYS (the reutilization of bases from dietary or catabolic sources) The biosynthesis of purine (A and G) begins with the synthesis of the ribose-phosphate Pentose phosphate pathway Ribose phosphate pyrophospho-KINASE The major regulatory step in purine biosynthesis is the conversion of PRPP to 5-Phosphoribosyl-1-amine * Glutamine PRPP Glutamate PPi Amidophosphoribosyl transferase Amidophosphoribosyl transferase is an important regulatory enzyme in purine biosynthesis. It is strongly inhibited by the end products IMP, AMP, and GMP. This type of inhibition is called FEEDBACK INHIBITION. Several amino acids are utilized in purine biosynthesis IMP is the precursor for both AMP and GMP, the base is also called hypoxanthine Conversion of Hypoxanthine to Adenine/Guanine. (N source) Aspartate (N source) Glutamine The common mechanistic theme for the conversion of A and G is the conversion of a carbonyl oxygen to an amino group Purines: where do the atoms come from? Purine intermediates include: 1. Glycine 2. 1 C units of 5,10 mTHF 3. Glutamine 4. Asparate The regulation of purine biosynthesis is a classic example of negative feedback Inhibited by AMP AMP Ribose 5-phosphate PRPP Phosphoribosyl amine IMP GMP Inhibited by IMP, AMP, and GMP Inhibited by GMP Stages of nucleotide metabolism Endonuclease Nucleic Acid Synthesis Phosphodiesterase Nucleoside monophosphates (Mononucleosides) Nucleoside triphosphate Endonuclease Nucleic Acid Synthesis Phosphodiesterase Nucleotidases H20 Nucleoside monophosphates (Mononucleosides) Pi Nucleoside triphosphate PPi ADP Nucleosides Nucleoside kinase ATP Phosphoribosyl transferases Pi PRPP Phosphorylases Ribose-1-P Nucleobases Uric Acid (purines) Salvage pathways for the re-utilization of purines; There are 2 salvage enzymes with different specificity; 1. Adenine phosphoribosyl transferase 2. Hypoxanthine-guanine phosphoribosyl transferase O O O P O CH2 OH OH O PPi + Base (ie Adenine) O OH P O CH2 OH OH A O OH A-PRT PRPP + Adenine Adenylate HG-PRT PRPP + Guanine Guanylate + PPi Purines in humans are degraded to Urate Important points: 1. Nucleotides are constantly undergoing turnover! 2. There are many enzymes involved; Nucleotidases Nucleoside phosphorylases Deaminases Xanthine oxidases 3. the final common intermediate in humans is Urate, which is excreted. 4. there are several metabolic disorders resulting from defects in purine catabolism. NH 2 HO 2 + 4 N N N N ribose O NH N HN Adenosine deaminase (ADA) ribose purine nucleoside phosphorylase N N H Hypoxanthine Inosine Adenosine N HN N N O ribose - 1 - P Pi xanthine oxidase Pi O N HN NH 2 ribose - 1 - P N N ribose O Guanosine N N H O + NH 4 N HN purine nucleoside NH 2 phosphorylase H 2O N HN Guanine deaminase O Xanthine Guanine xanthine oxidase O Catabolism of Purine Nucleotides N H N H HN O Uric acid O H N N H N H Disorders of Purine Metabolism: Disorder Gout Lesch Nyhan syndrome Defect PRPP synthase/ HGPRT lack of HGPRT Comments Hyperuricemia Hyperuricemia SCID ADA High levels of dAMP von Gierke’s disease glucose -6-PTPase Hyperuricemia a disorder associated with abnormal amounts of urates in the body early stage: recurring acute non-articular arthritis late stage: chronic deforming polyarthritis and eventual renal complication disease with rich history dating back to ancient Greece What happens in gout? Inhibited by AMP AMP Ribose 5-phosphate PRPP Phosphoribosyl amine IMP GMP Inhibited by IMP, AMP, and GMP Inhibited by GMP 1. Negative regulation of PRPP Synthatase & PRPP Amidotransferase is lost 2. PRPP levels are increased because of defects in salvage pathways Therefore, there is net increase in biosynthetic/degradation pathways!! GOUT (Gouty Arthritis): A defect of purine metabolism Serum Uric Acid Levels (mg/dl) Incidence of Gout (% of cases) >9.0 7-9 <7.0 ~10% 0.5-3.5% 0.1% Hypoxanthine Guanine xanthine oxidase Xanthine Urate xanthine oxidase Allopurinol: a. decrease urate b. increase xanthine & hypoxanthine c. decrease PRPP prevails mainly in adult males rarely encountered in premenopausal women symptoms are cause by deposition of crystals of monosodium urate monohydrate (can be seen under polarized light) usually affect joints in the lower extremities (the big toe is the classic site) SCID-Severe Combined Immunodeficiency Syndrome Autosomal recessive disorder Mutations in ADA AMP H20 Nucleotidase Pi Both T and B cells are significantly reduced (dATP is toxic) Adenosine H20 Adenine deaminase* NH3 Inosine Infants subject to bacterial, candidiasis, viral, protazoal infections Hypoxanthine 1995-AdV expressing ADA was successfully employed as gene therapy strategy Degradation of AMP OH NH2 NH3 H2 O N N N N AMP deaminase N N N N Ribose-P Ribose-P H2 O H2O Nucleotidase Nucleotidase Pi Pi O NH2 NH3 H2 O N N HN N Adenosine deaminase N N N Pi N Ribose Ribose-1-P Ribose Purine nucleoside phosphorylase O may be reused through salvage pathway N HN N N H hypoxanthine NH2 OH H2N N N N HN N N N N Ribose Ribose transition state H OH N HN N N O Ribose Pentostatin (a transition state analog) shows promise in combination with Ara-C (Vidarabine) N HN N N Ribose MECHANISM OF ADENOSINE DEAMINASE In the absence of ADA lymphocytes are destroyed deoxyadenosine is not destroyed, is converted to dAMP and then into dATP dATP is a potent feedback inhibitor of deoxynucleotide biosynthesis this leads to SCID (severe combined immunodeficiency disease) Infants with this deficiency have a high fatality rate due to infections treatment consists of administering pegylated ADA which can remain in the blood for 1 – 2 weeks more efficient is gene therapy: replacing the gene that is missing or defective gene therapy has been performed on selected patients Degradation of GMP and XMP O O H N N H2N N H N O N N Ribose-P N N H Ribose-P H2O H2O nucleotidase nucleotidase Pi Pi O O H N N N N N N H2N H O Pi N N Ribose PNP Ribose H Ribose-1P Pi PNP Ribose-1P O O H N N H N N H2N N N H H2O NH3 O N N H H http://www.wiley.com/college/fob/q uiz/quiz22/quizzer22.html Please, Send the online Quiz to my email; [email protected]