AIMST MBBS L30 Nucleic Acids - Chem Fn Metab PDF

AIMST UNIVERSITY FACULTY OF MEDICINE Bachelor of Medicine and Bachelor of Surgery (MBBS) MOLECULAR AND CELLULAR BASIS OF MEDICINE 1 INTRODUCTION TO BASIC SCIENCES L30 NUCLEIC ACIDS – CHEMISTRY, FUNCTION & METAB...

AIMST UNIVERSITY FACULTY OF MEDICINE Bachelor of Medicine and Bachelor of Surgery (MBBS) MOLECULAR AND CELLULAR BASIS OF MEDICINE 1 INTRODUCTION TO BASIC SCIENCES L30 NUCLEIC ACIDS – CHEMISTRY, FUNCTION & METABOLISM Prof. Dr. Abd. Rahman Md. Said Wed 23.10.24 10.00am MBBS L30 Nucleic acids LR1 20. 9. 2023 AIMST University Faculty of Medicine, MBBS.  Learning Objectives The objective of this lecture is to discuss: the structure of nitrogenous bases, the biological role of DNA and RNA and disorders associated. AIMST University Faculty of Medicine, BSc (Hons) Biomedical Sciences  Topic Outcomes 30.1 Describe the structure, function and biological role of nucleosides, nucleotides, and analogues. 30.2 Describe the structure, function, and types of nucleic acids (DNA and RNA). 30.3 Explain purine and pyrimidine biosynthetic pathways and its regulation. 30.4 Explain the role of ribonucleotide reductase. 30.5 Describe the disorders associated with purine and pyrimidine metabolism. AIMST University Faculty of Medicine, BSc (Hons) Biomedical Sciences  Nitrogenous Bases Planar, aromatic, and heterocyclic Derived from purine or pyrimidine Numbering of bases is “unprimed” 4 AIMST Univeersity, Jalan Bedong-08100, Kedah DA Sugars Pentoses (5-C sugars) Numbering of sugars is “primed” D-Ribose and 2’-Deoxyribose Lacks a 2’-OH group 5 AIMST Univeersity, Jalan Bedong-08100, Kedah DA Nucleosides Result from linking one of the sugars with a purine or pyrimidine base through an N-glycosidic linkage Purines bond to the C1’ carbon of the sugar at their N9 atoms Pyrimidines bond to the C1’ carbon of the sugar at their N1 atoms AIMST Univeersity, Jalan Bedong-08100, Kedah DA 6 Nucleotides Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through esterification. Phosphates can be bonded to either C3 or C5 atoms of the sugar. Mono-, di- or triphosphates. AIMST Univeersity, Jalan Bedong-08100, Kedah DA 7 Nucleotides Monomers for nucleic acid polymers Nucleoside Triphosphates are important energy carriers (ATP, GTP) Important components of coenzymes FAD, NAD+ and Coenzyme A RNA (ribonucleic acid) is a polymer of ribonucleotides DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides Both deoxy- and ribonucleotides contain Adenine, Guanine and Cytosine Ribonucleotides contain Uracil Deoxyribonucleotides contain Thymine AIMST Univeersity, Jalan Bedong-08100, Kedah DA 8 Naming Conventions Nucleosides: Purine nucleosides end in “-sine” Adenosine, Guanosine Pyrimidine nucleosides end in “-dine” Thymidine, Cytidine, Uridine Nucleotides: Start with the nucleoside name from above and add “mono-”, “di-”, or “triphosphate” Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate AIMST Univeersity, Jalan Bedong-08100, Kedah DA 9 Nucleotide Metabolism PURINE RIBONUCLEOTIDES: formed de novo i.e., purines are not initially synthesized as free bases First purine derivative formed is Inosine Mono-phosphate (IMP) The purine base is hypoxanthine AMP and GMP are formed from IMP AIMST Univeersity, Jalan Bedong-08100, Kedah DA 10 Purine Nucleotides Purine ring components came from: N1: Aspartate Amine C2, C8: Formate N3, N9: Glutamine C4, C5, N7: Glycine C6: Bicarbonate Ion AIMST Univeersity, Jalan Bedong-08100, Kedah DA 11 Purine Nucleotide Synthesis AIMST Univeersity, Jalan Bedong-08100, Kedah DA 12 Purine Nucleotide Synthesis ATP is involved in 6 steps PRPP in the first step of Purine synthesis is also a precursor for Pyrimidine Synthesis, His and Trp synthesis Role of ATP in first step is unique– group transfer rather than coupling In second step, C1 notation changes from α to β (anomers specifying OH positioning on C1 with respect to C4 group) In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” stHydrolyzing a phosphate from ATP is relatively easy ΔG°’= -30.5 kJ/mol If endergonic reaction released energy into cell as heat energy, wouldn’t be useful Must be coupled to an exergonic reaction When ATP is a reactant: Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl, or adenosinyl group ATP hydrolysis can drive an otherwise unfavorable reaction (synthetase; “energase”) AIMST Univeersity, Jalan Bedong-08100, Kedah DA 13 Purine Biosynthetic Pathway Channeling of some reactions on pathway organizes and controls processing of substrates to products in each step Increases overall rate of pathway and protects intermediates from degradation In animals, IMP synthesis pathway shows channeling at: Reactions 3, 4, 6 Reactions 7, 8 Reactions 10, 11 AIMST Univeersity, Jalan Bedong-08100, Kedah DA 14 IMP Conversion to AMP AIMST Univeersity, Jalan Bedong-08100, Kedah DA 15 IMP Conversion to GMP AIMST Univeersity, Jalan Bedong-08100, Kedah DA 16 Regulatory Control of Purine Nucleotide Biosynthesis GTP is involved in AMP synthesis and ATP is involved in GMP synthesis (reciprocal control of production) PRPP is a biosynthetically “central” molecule (why?) ADP/GDP levels – negative feedback on Ribose Phosphate Pyrophosphokinase Amidophosphoribosyl transferase is activated by PRPP levels APRT activity has negative feedback at two sites ATP, ADP, AMP bound at one site GTP,GDP AND GMP bound at the other site Rate of AMP production increases with increasing concentrations of GTP; rate of GMP production increases with increasing concentrations of ATP AIMST Univeersity, Jalan Bedong-08100, Kedah DA 17 Regulatory Control of Purine Biosynthesis Above the level of IMP production: Independent control Synergistic control Feedforward activation by PRPP Below level of IMP production Reciprocal control Total amounts of purine nucleotides controlled Relative amounts of ATP, GTP controlled AIMST Univeersity, Jalan Bedong-08100, Kedah DA 18 Purine Catabolism and Salvage All purine degradation leads to uric acid (but it might not stop there) Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases in the intestine Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosides Direct absorption of nucleosides Further degradation Nucleoside + H2O 🡪 base + ribose (nucleosidase) Nucleoside + Pi 🡪 base + r-1-phosphate (n. phosphorylase) NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETED. AIMST Univeersity, Jalan Bedong-08100, Kedah DA 19 Intracellular Purine Catabolism Nucleotides broken into nucleosides by action of 5’-nucleotidase (hydrolysis reactions) Purine nucleoside phosphorylase (PNP) Inosine 🡪 Hypoxanthine Xanthosine 🡪 Xanthine Guanosine 🡪 Guanine Ribose-1-phosphate splits off Can be isomerized to ribose-5-phosphate Adenosine is deaminated to Inosine (ADA) AIMST Univeersity, Jalan Bedong-08100, Kedah DA 20 Intracellular Purine Catabolism Xanthine is the point of convergence for the metabolism of the purine bases Xanthine 🡪 Uric acid Xanthine oxidase catalyzes two reactions Purine ribonucleotide degradation pathway is same for purine deoxyribonucleotides AIMST Univeersity, Jalan Bedong-08100, Kedah DA 21 Adenosine Degradation AIMST Univeersity, Jalan Bedong-08100, Kedah DA 22 Xanthosine Degradation Ribose sugar gets recycled (Ribose-1-Phosphate 🡪 R-5-P ) – can be incorporated into PRPP (efficiency) Hypoxanthine is converted to Xanthine by Xanthine Oxidase Guanine is converted to Xanthine by Guanine Deaminase Xanthine gets converted to Uric Acid by Xanthine Oxidase AIMST Univeersity, Jalan Bedong-08100, Kedah DA 23 Xanthine Oxidase A homodimeric protein Contains electron transfer proteins FAD Mo-pterin complex in +4 or +6 state Two 2Fe-2S clusters Transfers electrons to O2 🡪 H2O2 H2O2 is toxic Disproportionated to H2O and O2 by catalase AIMST Univeersity, Jalan Bedong-08100, Kedah DA 24  THE PURINE NUCLEOTIDE CYCLE AMP + H2O 🡪 IMP + NH4+ (AMP Deaminase) IMP + Aspartate + GTP 🡪 AMP + Fumarate + GDP + Pi (Adenylosuccinate Synthetase) COMBINE THE TWO REACTIONS: Aspartate + H2O + GTP 🡪 Fumarate + GDP + Pi + NH4+ The overall result of combining reactions is deamination of Aspartate to Fumarate at the expense of a GTP AIMST Univeersity, Jalan Bedong-08100, Kedah DA 25 Uric Acid Excretion Humans – excreted into urine as insoluble crystals Birds, terrestrial reptiles, some insects – excrete insoluble crystals in paste form Excess amino N converted to uric acid (conserves water) Others – further modification : Uric Acid 🡪 Allantoin 🡪 Allantoic Acid 🡪 Urea 🡪 Ammonia AIMST Univeersity, Jalan Bedong-08100, Kedah DA 26 Purine Salvage Adenine phosphoribosyl transferase (APRT) Adenine + PRPP 🡪 AMP + PPi Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT) Hypoxanthine + PRPP 🡪 IMP + PPi Guanine + PRPP 🡪 GMP + PPi (NOTE: THESE ARE ALL REVERSIBLE REACTIONS) AMP,IMP,GMP do not need to be resynthesized de novo ! AIMST Univeersity, Jalan Bedong-08100, Kedah DA 27 Gout Impaired excretion or overproduction of uric acid Uric acid crystals precipitate into joints (Gouty Arthritis), kidneys, ureters (stones) Lead impairs uric acid excretion – lead poisoning from pewter drinking goblets Fall of Roman Empire? Xanthine oxidase inhibitors inhibit production of uric acid, and treat gout Allopurinol treatment – hypoxanthine analog that binds to Xanthine Oxidase to decrease uric acid production AIMST Univeersity, Jalan Bedong-08100, Kedah DA 28 ALLOPURINOL IS A XANTHINE OXIDASE INHIBITOR A SUBSTRATE ANALOG IS CONVERTED TO AN INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR” AIMST Univeersity, Jalan Bedong-08100, Kedah DA 29 Lesch-Nyhan Syndrome A defect in production or activity of HGPRT Causes increased level of Hypoxanthine and Guanine (🡪↑ in degradation to uric acid) Also,PRPP accumulates stimulates production of purine nucleotides (and thereby increases their degradation) Causes gout-like symptoms, but also neurological symptoms 🡪 spasticity, aggressiveness, self-mutilation First neuropsychiatric abnormality that was attributed to a single enzyme AIMST Univeersity, Jalan Bedong-08100, Kedah DA 30 Purine Autism 25% of autistic patients may overproduce purines To diagnose, must test urine over 24 hours Biochemical findings from this test disappear in adolescence Must obtain urine specimen in infancy, but it’s difficult to do! Pink urine due to uric acid crystals may be seen in diapers AIMST Univeersity, Jalan Bedong-08100, Kedah DA 31 Pyrimidine Ribonucleotide Synthesis Uridine Monophosphate (UMP) is synthesized first CTP is synthesized from UMP Pyrimidine ring synthesis completed first; then attached to ribose-5-phosphate N1, C4, C5, C6 : Aspartate C2 : HCO3- N3 : Glutamine amide Nitrogen AIMST Univeersity, Jalan Bedong-08100, Kedah DA 32 Pyrimidine Synthesis AIMST Univeersity, Jalan Bedong-08100, Kedah DA 33  UMP Synthesis Overview 2 ATPs needed: both used in first step One transfers phosphate, the other is hydrolyzed to ADP and Pi 2 condensation rxns: form carbamoyl aspartate and dihydroorotate (intramolecular) Dihydroorotate dehydrogenase is an intra-mitochondrial enzyme; oxidizing power comes from quinone reduction Attachment of base to ribose ring is catalyzed by OPRT; PRPP provides ribose-5-P PPi splits off PRPP – irreversible Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on same chain AIMST Univeersity, Jalan Bedong-08100, Kedah DA 34 OMP DECARBOXYLASE : THE MOST CATALYTICALLY PROFICIENT ENZYME FINAL REACTION OF PYRIMIDINE PATHWAY ANOTHER MECHANISM FOR DECARBOXYLATION A HIGH ENERGY CARBANION INTERMEDIATE NOT NEEDED NO COFACTORS NEEDED ! SOME OF THE BINDING ENERGY BETWEEN OMP AND THE ACTIVE SITE IS USED TO STABILIZE THE TRANSITION STATE “PREFERENTIAL TRANSITION STATE BINDING” AIMST Univeersity, Jalan Bedong-08100, Kedah DA 35 UMP 🡪 UTP and CTP Nucleoside monophosphate kinase catalyzes transfer of Pi to UMP to form UDP; nucleoside diphosphate kinase catalyzes transfer of Pi from ATP to UDP to form UTP CTP formed from UTP via CTP Synthetase driven by ATP hydrolysis Glutamine provides amide nitrogen for C4 in animals AIMST Univeersity, Jalan Bedong-08100, Kedah DA 36 AIMST Univeersity, Jalan Bedong-08100, Kedah DA 37  Regulatory Control of Pyrimidine Synthesis Differs between bacteria and animals Bacteria – regulation at ATCase rxn Animals – regulation at carbamoyl phosphate synthetase II UDP and UTP inhibit enzyme; ATP and PRPP activate it UMP and CMP competitively inhibit OMP Decarboxylase *Purine synthesis inhibited by ADP and GDP at ribose phosphate pyrophosphokinase step, controlling level of PRPP 🡪 also regulates pyrimidines AIMST Univeersity, Jalan Bedong-08100, Kedah DA 38 Degradation of Pyrimidines CMP and UMP degraded to bases similarly to purines Dephosphorylation Deamination Glycosidic bond cleavage Uracil reduced in liver, forming β-alanine Converted to malonyl-CoA 🡪 fatty acid synthesis for energy metabolism AIMST Univeersity, Jalan Bedong-08100, Kedah DA 39 Deoxyribonucleotide Formation Purine/Pyrimidine degradation are the same for ribonucleotides and deoxyribonucleotides Biosynthetic pathways are only for ribonucleotide production Deoxyribonucleotides are synthesized from corresponding ribonucleotides AIMST Univeersity, Jalan Bedong-08100, Kedah DA 40 ADENOSINE DEAMINASE DEFICIENCY IN PURINE DEGRADATION, ADENOSINE 🡪 INOSINE ENZYME IS ADA ADA DEFICIENCY RESULTS IN SCID “SEVERE COMBINED IMMUNODEFICIENCY” SELECTIVELY KILLS LYMPHOCYTES BOTH B- AND T-CELLS MEDIATE MUCH OF IMMUNE RESPONSE ALL KNOWN ADA MUTANTS STRUCTURALLY PERTURB ACTIVE SITE 41 AIMST Univeersity, Jalan Bedong-08100, Kedah DA Adenosine Deaminase CHIME Exercise: 2ADA Enzyme catalyzing deamination of Adenosine to Inosine α/β barrel domain structure “TIM Barrel” – central barrel structure with 8 twisted parallel β-strands connected by 8 α-helical loops Active site is at bottom of funnel-shaped pocket formed by loops Found in all glycolytic enzymes Found in proteins that bind and transport metabolites AIMST Univeersity, Jalan Bedong-08100, Kedah DA 42  References 1. Harper’s Illustrated Biochemistry; 26th edition, ch13, pg 102 to 110 2. Google images 3. Google scholar 4. http://www.webmd.com/diet/fiber-health-benefits-11/insoluble-soluble-fiber?pag e=1 5. TEXTBOOK OF BIOCHEMISTRY, for Medical Students Sixth Edition by DM Vasudevan 6. McGraw-Hills AccessMedicine. 7. http://www.nbs.csudh.edu/chemistry/faculty/nsturm/CHE452/19_Thyroid15.htm 8. Labtestsonline AIMST Univeersity, Jalan Bedong-08100, Kedah DA 43 THANK YOU AIMST Univeersity, Jalan Bedong-08100, Kedah DA 44