Chapter 10 Metabolism of Nucleotides For 2 ndyear
Chapter 10 Metabolism of Nucleotides For 2 nd-year Medical Students Guangchao Liu , Ph. D Department of Biochemistry Faculty of Medicine Henan University 目 录
Overview 1. Digestion and Absorption of Nucleotides and Nucleic Acids l Nucleoprotein (in food) Stomach Acid Nucleic Acids (RNA & DNA) Protein Nuleases Nucleotides Nucleosides Base Nucleotidases Phosphate Nucleosides Pentose 目 录
Nucleoproteins Dietary nucleoprotein Pancreatic enzymes Tissue nucleoprotein Lysosomal enzymes Metabolism 目 录
Nucleic acids (endonucleases) (oligonucleotide) 目 录
Ingested nucleic acids and nucleotides may be absorbed or converted to purine and pyrimidine bases. The purine bases are then oxidized to uric acid, which may be absorbed and excreted in the urine. While little or no dietary purine or pyrimidine is incorporated into tissue nucleic acids, injected compounds are incorporated. 目 录
• 2. Funcions of Nucleotides 1. They are the precursors of DNA and RNA. 2. They are essential carriers of chemical energy—a role primarily of ATP and to some extent GTP. 3. They are components of the cofactors NAD, FAD, Sadenosylmethionine, and coenzyme A. 4. Nucleotide derivatives are activated intermediates in biosynthetic processes (UDPG, SAM) 5. Serve as metabolic regulators (e. g ADP and ATP). 6. such as c. AMP and c. GMP, are also cellular second messengers 7. Nucleotides play key roles in many cellular processes 目 录
Segment 1. Metabolism of Purine Nucleotides 目 录
l The structures of Purine Nucleotides AMP GMP 目 录
1. 1 Anabolism of Purine Nucleotides l de novo synthesis pathway • De novo synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose 5 -phosphate, CO 2, and NH 3. • Salvage synthesis pathway • Salvage pathways recycle the free bases and nucleosides released from nucleic acid breakdown. 目 录
PURINES & PYRIMIDINES ARE DIETARILY NONESSENTIAL Ingested nucleic acids and nucleotides, are dietarily nonessential, Human tissues can synthesize purines and pyrimidines from amphibolic intermediates. 目 录
1. 1. 1 de novo anabolism pathway Ø Their metabolic precursors: amino Asp、Gly、Gln、CO 2、Ribose 5 -phosphate 、 Formate Ø The tissues or organs • Most of the de novo synthesis of the bases of nucleotides occurs in the liver, and to some extent in the brain, neutrophils, and other cells of the immune system. • (Subcellular:Cytosol) Ø Process 1. IMP synthesis 2. IMP Conversion to AMP &GMP IMP Conversion to 目 录
Purine ribonucleotides synthesis: The initially synthesized purine derivative is inosine monophosphate (IMP); IMP is the precursor of both AMP and GMP Highly divergent organisms have very similar pathways. 目 录
• Origin of the ring atoms • of purine bases 目 录
The biosynthesis of purine (A and G) begins with the synthesis of the ribose-phosphate Pentose phosphate pathway Ribose phosphate pyrophospho-KINASE 目 录
1) IMP Synthesis 目 录
Purine Nucleotide Synthesis at a Glance 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 n Role of ATP in first step is unique– group transfer rather than coupling In second step, C 1 notation changes from a to b (anomers specifying OH positioning on C 1 with respect to C 4 group) In step 3, PPi is hydrolyzed to 2 Pi (irreversible, “committing” step) 目 录
Multifunctional Catalysts Participate in urine Nucleotide Biosynthesis In prokaryotes, each reaction is catalyzed by a different polypeptide. By contrast, in eukaryotes, the enzymes are polypeptides with multiple catalytic activities whose adjacent catalytic sites facilitate channeling of intermediates between sites. Three distinct multifunctional enzymes catalyze reactions 3, 4, and 6, reactions 7 and 8, and reactions 10 and 11 目 录
Purines are initially formed as ribonucleotides IMP rather than as free bases. Purine ring synthesis requires 11 steps leading to production of inosinate (IMP) many precursors: 2 glutamine, 1 glycine, 1 CO 2, 1 aspartate, 2 formate energy expenditure: 5 ATP 目 录
2) IMP Conversion to AMP & GMP 目 录
Nucleoside Monophosphates Are Converted to Nucleoside diphosphates &Nucleoside Triphosphates The conversion pathways are common to all cells. Enzymes: adenosine 5'-diphosphate(ADP) &Guanosine 5'diphosphate(GDP) form : adenylate kinase & guanylate kinase Nucleotide triphosphate (ATP & GTP) form : nucleotide diphosphokinase 目 录
AMP Adenylate kinase Guanylate kinase ADP ATP ATP Nucleotide diphosphokinase GMP GDP ATP ADP Nucleotide diphosphokinase ATP GTP ADP 目 录
Regulatory Control of Purine Nucleotide Biosynthesis 目 录
• Regulatory mechanisms • Purine Nucleotide Biosynthesis Is Regulated _ _) or positive (+ ) feedback by negative (– _ + regulation R-5 -P ATP + synthetase amidotransferase _PRA PRPP _ IMP Adenylo AMP ADP ATP succinate XMP GDP GTP _ Adenylo AMP + IMP GTPsuccinate XMP ATP +GMP ADP ATP GDP GTP _ 目 录
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? ) n n n ADP/GDP levels – negative feedback on Ribose Phosphate Pyrophosphokinase Amidophosphoribosyl transferase is activated by PRPP levels APRT activity has negative feedback at two sites w ATP, ADP, AMP bound at one site w 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 目 录
Regulatory Control of Purine Biosynthesis At level of IMP production: n n n Independent control Synergistic control Feedforward activation by PRPP Below level of IMP production n Reciprocal control Total amounts of purine nucleotides controlled Relative amounts of ATP, GTP controlled 目 录
1. 1. 2 The salvage pathways of Purine nucleotide Conversion of purines, their ribonucleosides, and their deoxyribonucleosides to mononucleotides Free purines are in large part salvaged and reused to make mononucleotides, in a pathway much simpler than the de novo synthesis of purine nucleotides described earlier. 目 录
• Their metabolic precursors: purines、purine nucleosides、PRPP • Tissues and rogans: Bone marrow、brain。 • Enzymes: adenine phosphoribosyl transferase, APRT hypoxanthine- guanine phosphoribosyl transferase, HGPRT adenosine kinase 目 录
• Process A + PRPP I + PRPP G + PRPP adenosine APRT HGPRT AMP + PPi IMP + PPi GMP + PPi adenosine kinase AMP ATP ADP • The importance Energy saving process Relied upon more heavily by some tissues than others 目 录
Lesch-Nyhan syndrome 1. Michael Lesch was a medical student at Johns Hopkins Hospital, . 2 pediatrician William Leo Nyhan was a faculty member, when the two identified LNS and its associated hyperuricemia in two affected brothers )aged four and eight(, and published their findings. LNS patients http//: images. google. co. th/imgres? imgurl=http//: www. webst. it/leschnyhan/simone. jpg&imgrefurl=http//: www. webst. it/leschnyhan/engl 2. htm&h=188&w=162&sz=15&tbnid=PYog. Qs. PQf. Huh. YM: &tbnh=97&tbnw=83&hl=th&start=6&prev/=images 目 录 %3 Fq%3 DLesch. Nyhan%2 Bsyndrome%2 Band%2 Bpictures%26 svnum%3 D 10%26 hl%3 Dth%26 lr%3 D%26 sa%3 DX
Lesch-Nyhan Syndrome: HGPRT Deficiency Leads to Severe Clinical Disorder Defect in HGPRT Prevents effective salvaging of purines Compulsive self-destructive behavior Mental deficiency Bite off fingers and lips Aggression Sex-linked, single gene defect Markers: increased PRPP increased de novo purine production increased urate production 目 录
1. 2 The conversion of Purine nucleotides + H + H AMP P D A N Ad e no si ne Adenylo succinate de am i NH 3 na s + P D A N e IMP NH 3 P GM GMP e as c du re XMP w KEY:nucleosine monophosphate Level 目 录
The purine nucleotide cycle This pathway functions in muscle to prime the citric acid cycle by generating fumarate. 目 录
1. 3 Biosynthesis of deoxyribonucleotides Deoxyribonucleotides are synthesized from corresponding ribonucleotides by reduction of their C 2’ position rather than by their de novo synthesis from deoxyribose-containing precursors. ribonucleotide reductase 目 录
Formation of deoxyribonucleotides Note: nucleosine monophosphate Level (N: A、G、U、C) 目 录
Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase. • Electrons are transmitted (blue arrows) to the enzyme from NADPH by (a) glutaredoxin or (b) thioredoxin. The sulfide groups in glutaredoxin reductase are contributed by two molecules of bound glutathione (GSH; GSSG indicates oxidized NADPH provides the reducing equivalents for this process through the intermediacy glutathione). 目 录
Regulation of 2′deoxyribonucleotides synthesis. 目 录
Enzymatic mechanism of ribonucleotide reductase Class I ribonucleotide reductase from E. coli: 目 录
Regulation of ribonucleotide reductase by deoxynucleoside triphosphates. 目 录
1. 4 metabolic antagonist of purine nucleotide • Analog of Purine 、Amino Acid or Folic Acid。 • A growing array of important chemotherapeutic agents—for cancer and other diseases—act by target-inhibiting one or more enzymes in the pathways. purine analogues 6 -Mercaptopurine 6 -thioguanine 8 -azaguanine Allopurinol amino acid analogues Azaserine Acivicin diazonorleucin e,DAL Folic acid analogues aminopterin Methotrexate Trimethoprim 目 录
Anticancer drugs Antifolate : folic acid analogue trimethoprim 目 录
2 Purine catabolism: to uric acids The various nucleotides are first converted to nucleosides by intracellular nucleosidase. Adenoside and deoxyadenosine are first converted to inosine by adenosine deaminase and then to hyproxanthine by purine nucleoside phosphorylase (NPN). The PNP products are merged into xanthine by guanine deaminase and xanthine oxidase, and xanthine is then oxidized to uric acid by this latter enzyme. 目 录
2. 1 Nucleotide Degredation: The major pathways of purine catabolism in animals Adenosine deaminase PDBid 1 ADA See Interactive Exercises 目 录
Xanthine oxidase is a mini-electron-transport system Xanthine oxidase (XO) converts hypoxanthine (IMP base) to xanthine, and xanthine to uric acid--reaction product is an enol with p. K of 5. 4. the enol tautomerizes to the more stable keto form. 目 录
2. 2 Gout is caused by an excess of uric acid which leads to deposition of nearly insoluble crystals of sodium urate. Sodium urate and/or uric acid may also precipitate in kidneys and ureters as stones. 目 录
Gout therapy 目 录
Purine Autism 25% of autistic patients may overproduce purines To diagnose, must test urine over 24 hours n n Biochemical findings from this test disappear in adolescence Must obtain urine specimen in infancy, but it’s difficult to do! w Pink urine due to uric acid crystals may be seen in diapers 目 录
Severe combined immunodeficiency disease (SCID) 目 录
ADENOSINE DEAMINASE DEFICIENCY IN PURINE DEGRADATION, ADENOSINE INOSINE n ENZYME IS ADA DEFICIENCY RESULTS IN SCID n “SEVERE COMBINED IMMUNODEFICIENCY” SELECTIVELY KILLS LYMPHOCYTES n n BOTH B- AND T-CELLS MEDIATE MUCH OF IMMUNE RESPONSE ALL KNOWN ADA MUTANTS STRUCTURALLY PERTURB ACTIVE SITE 目 录
Adenosine Deaminase CHIME Exercise: 2 ADA Enzyme catalyzing deamination of Adenosine to Inosine a/b barrel domain structure n “TIM Barrel” – central barrel structure with 8 twisted parallel b-strands connected by 8 a-helical loops n Active site is at bottom of funnel-shaped pocket formed by loops n Found in all glycolytic enzymes n Found in proteins that bind and transport metabolites 目 录
Segment 2. Metabolism of Pyrimidine Nucleotides 目 录
The structures of pyrimidine Nucleotides l 目 录
1. Pyrimidine anabolism pathway l Pyrimidine anabolism de novo pathway • Pyrimidine Nucleotides Are Made from Aspartate, PRPP, and Carbamoyl Phosphate l Pyrimidine salvage anabolism pathway Salvage pathways recycle the free bases and nucleosides released from nucleic acid breakdown. 目 录
1. 1 Pyrimidine anabolism de novo pathway Ø precursor Asp、Gln、CO 2、5 -P-R Process: Mainly in liver cell cytoplasm 目 录
ATP, PRPP carbamoyl phosphate synthetase II (gln) Glutamine + 2 ATP + CO 2 - UDP UTP Glu + Pi + ADP CTP Glu + Pi + 2 ADP Gln + ATP +2 ADP UTP carbamoyl phosphate aspartate transcarbamoylase carbamoylaspartate +2 ATP orotate phosphoribosyl transferase UMP OMP +PRPP orotidylic acid decarboxylase Figure 5. Biosynthesis of the pyrimidine nucleotides UTP and CTP. 目 录
1. 1. 1 de novo pathway In cytoplasm * 目 录
The catalyst for the initial reaction is cytosolic carbamoyl phosphate synthase II, a different enzyme from the mitochondrial carbamoyl phosphate synthase I of urea synthesis 目 录
UMP Synthesis 目 录
Multifunctional Proteins Catalyze the Early Reactions of Pyrimidine Biosynthesis Five of the first six enzyme activities of pyrimidine biosynthesis reside on multifunctional polypeptides. One such polypeptide catalyzes the first three reactions 1, 2 & 3 of Figure and ensures efficient channeling of carbamoyl phosphate to pyrimidine biosynthesis. A second bifunctional enzyme catalyzes reactions 5 and 6. 目 录
Most of enzymes in cytosol,some one in mitochondrion First, pyrimidine ring is synthezed ; 目 录
CTP & TMP Synthesis 目 录
d. TMP Synthesis ribonucleotide UDP reductase d. UDP CTP d. CMP CDP d. CDP TMP synthase N 5, N 10 -甲烯FH 4 FH 2 reducase NADP+ d. UMP FH 2 NADPH+H+ d. TMP 目 录
• 1. 1. 2 Regulatory Control of Pyrimidine Synthesis ATP + CO 2+ GLn - + carbomoyl phosphate Asp carbamyl aspartate PRPP - - UMP UTP _ TDP ATP + 5 -P-R AMP & GMP CTP 目 录
Regulatory Control of Pyrimidine Synthesis Differs between bacteria and animals n Bacteria – regulation at ATCase rxn Animals – regulation at carbamoyl phosphate synthetase II n n 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 目 录
Purine & Pyrimidine Nucleotide Biosynthesis Are Coordinately Regulated Purine and pyrimidine biosynthesis parallel one another mole for mole, suggesting coordinated control of their biosynthesis. Several sites of cross-regulation characterize purine and pyrimidine nucleotide biosynthesis. The PRPP synthase reaction is feedback-inhibited by both purine and pyrimidine nucleotides. 目 录
Orotic Aciduria Caused by defect in protein chain with enzyme activities of last two steps of pyrimidine synthesis Increased excretion of orotic acid in urine Symptoms: retarded growth; severe anemia Only known inherited defect in this pathway (all others would be lethal to fetus) Treat with uridine/cytidine IN-CLASS QUESTION: HOW DOES URIDINE AND CYTIDINE ADMINISTRATION WORK TO TREAT OROTIC ACIDURIA? 目 录
1. 2. Pyrimidine anabolism : salvage pathway ATPdependentphosphoryltransferases (kinases( orotate phosphoribosyltransferase U + PRPP uridine + ATP Thymidine + ATP • Uracil phosphoribosyl UMP+PPi transferase (UPRTase) Uridine kinase UMP+ADP Thymidine kinase TMP+ADP THE DEOXYRIBONUCLEOSIDES OF U & C ARE SALVAGED 目 录
2. The major pathways of pyrimidine catabolism in animals The amino acid products of these reactions are taken up in other metabolic processes. UMP and d. TMP are degraded by the same enzymes; the pathway for d. TMP degradation is given in parentheses. 目 录
Pyrimidine catabolism II b-alanine Acetyl Co. A NH 3 CO 2 b-amino-isobutyrate Succinyl Co. A NH 3 CO 2 目 录
3 metabolic antagonist of Pyrimidine nucleotide • Analog of Pyrimidine 、Amino Acid or Folic Acid。 • A growing array of important chemotherapeutic agents—for cancer and other diseases—act by target-inhibiting one or more enzymes in the pathways. T 5 -FU 目 录
Cytarabine Cyclocytidine 目 录
Anticancer drugs 5 -fluorouracil 5 -fluorodeoxyuridine 5 -fluorodeoxyuridylate, Fd. UMP Inhibit thymidylate synthase Inhibit DNA synthesis 目 录
Mechanism of action of 5 -FU 目 录 http//: www. xeloda. com/hcp/mechanism-of-action/default. aspx
TTP UTPase antifolate Dihydrofolate reductase + NADPH TTP Tetrahydrofolate (THF) dihydrofolate TDP diphosphokinase d. UTP fluorouridine Thymidylate synthetase + Vit. B 12 TMP kinase TDP 目 录
Magaloblastic anemia Chronic B 12 deficiency causes anemia: Megaloblastic anemia 目 录
Thank you! 目 录
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