BIOL214 Nucleotides I Notes PDF

BIOL214: Biochemistry of Energy and Metabolism Nucleotides Part I Prof. Ronald Sluyter Subject Coordinator [email protected] Lehninger Principles of Biochemistry, 8e, Chapters 8 and 22.4...

BIOL214: Biochemistry of Energy and Metabolism Nucleotides Part I Prof. Ronald Sluyter Subject Coordinator [email protected] Lehninger Principles of Biochemistry, 8e, Chapters 8 and 22.4 1  Learning Outcomes Recall the basic structure of nucleotides Describe the de novo synthesis of purine nucleotides and its regulation Describe the de novo synthesis of pyrimidine nucleotides and its regulation Describe how ribonucleotides form deoxyribonucleotides Describe examples relating to nucleotide synthesis and disease 2  Nucleotides have Important Biological Functions Storage of genetic information: DNA and RNA Chemical energy: ATP, UTP, GTP and CTP Components of major coenzymes: Coenzyme A, nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) Regulation of cell metabolism: e.g. AMP and phosphorylation Intracellular signalling: e.g. Cyclic AMP (cAMP) and phosphorylation Extracellular signalling: Adenosine and P1 receptors, and ATP (and other nucleotides) and P2 receptors 3 Nucleotide Structure: Revision Glycosidic linkage Glycosidic linkage -OH group in ribonucleotide -H group in deoxyribonucleotide 4 Major Purine and Pyrimidine Bases of Nucleic Acids : Revision 5  Nucleotide Nomenclature: Revision Base Nucleoside Nucleotide Nucleic acid Purines Adenine Adenosine Adenylate (AMP) RNA Deoxyadenosine Deoxyadenylate (dAMP) DNA Guanine Guanosine Guanylate RNA Deoxyguanosine Deoxyguanylate DNA Pyrimidines Cytosine Cytidine Cytidylate RNA Deoxycytidine Deoxycytidylate DNA Thymine Thymidine or deoxythymidine Thymidylate or DNA deoxythymidylate Uracil Uridine Uridylate RNA 6  Nucleotide Nomenclature: Revision N = nucleoside 7  Learning Outcomes Recall the basic structure of nucleotides Describe the de novo synthesis of purine nucleotides and its regulation Describe the de novo synthesis of pyrimidine nucleotides and its regulation Describe how ribonucleotides form deoxyribonucleotides Describe examples relating to nucleotide synthesis and disease 8  Two Pathways Lead to Nucleotides De novo pathways: Begin with metabolic precursors - ribose 5-phosphate, amino acids, CO2 and NH3 - and is metabolically expensive – Bases are synthesized while attached to ribose – Pyrimidine ring is synthesised as orotate – Glutamine provides most amino groups – Glycine is the precursor for purines – Aspartate is the precursor for pyrimidines Salvage pathways: Recycles the free bases and nucleosides released from nucleic acid breakdown and is metabolically economical (next lecture) 9 Origin of Ring Atoms in Purines 10  De Novo Purine Synthesis Begins with PRPP Purine ring assembled, atom by atom, on 5- phosphoribosyl 1-pyrophospate (PRPP) (Activated ribose) Glutamine-PRPP In the first committed step, an amino group amidotransferase donated by glutamine is attached at C-1 of PRPP to form 5-phosphoribosylamine In the second step, three atoms are added from glycine, requiring ATP A further eight steps are required in higher eukaryotes to form the ring structure and the nucleotide inosinate (IMP) (nine steps in bacteria; see Figure 22-35 for all 10-11 steps) 11  De Novo Purine Synthesis Results in the First Complete Purine Inosinate (IMP) In the tenth step, a second ring closure occurs to form inosinate (IMP) IMP is the first intermediate with a complete purine ring * In addition to its central role in purine synthesis, it is used as a flavour enhancer (E631) in a variety of foods *6a Alternative path from AIR to CAIR in higher eukaryotes (bypasses steps 6 and 7 as used by bacteria) 12  Inosinate (IMP) Forms a Branch Point in De Novo Purine Synthesis Conversion of IMP to AMP requires an amino group from aspartate, requiring GTP Conversion of IMP to GMP requires an amino group from glutamine, requiring ATP 13  Purine Biosynthesis is Regulated by Feedback Inhibition Four major feedback mechanisms cooperate in regulating the: 4 GDP from GMP also – Overall rate of de novo purine nucleotide Activated ribose impairs this step synthesis 1 – Relative rates of AMP and GMP synthesis From previous 3 slide 2 2 3 This reciprocal regulation balances the synthesis of each ribonucleotide 14  Revision What three molecules comprise a nucleotide? Which name is adenylate also known as? Name the two pathways by which nucleotides are formed. Complete the sentence. _______ is an important branch point in de novo purine synthesis. Outline the first committed step in de novo purine synthesis. Name the enzyme that catalyses and the nucleotides that impair this first committed step. Name the amino acids required to form AMP and GMP from IMP. True or false. GTP and ATP impair AMP and GMP synthesis, respectively. 15  Learning Outcomes Recall the basic structure of nucleotides Describe the de novo synthesis of purine nucleotides and its regulation Describe the de novo synthesis of pyrimidine nucleotides and its regulation Describe how ribonucleotides form deoxyribonucleotides Describe examples relating to nucleotide synthesis and disease 16  Pyrimidine Nucleotides are Made from Aspartate, PRPP and Carbamoyl Phosphate Pyrimidine synthesis proceeds by first making the Orotate pyrimidine ring (in the form of orotate) Aspartate and carbamoyl phosphate provide the atoms for the ring structure, making synthesis pyrimidine ring simpler than purine ring Ribose 5-phosphate is attached after pyrimidine ring formation Carbamoyl Aspartate phosphate 17  Carbamoyl Phosphate Synthetase II Cytosolic enzyme that makes the carbamoyl phosphate required in pyrimidine biosynthesis (know this enzyme) Different from carbamoyl phosphate synthetase I in the mitochondria required in urea cycle (see Urea Cycle lecture later in Session) Catalyses: 2 ATP + L-glutamine + HCO3- → 2 ADP + Pi + L-glutamate + carbamoyl phosphate To balance purine:pyrimidine IMP 18 Purine synthesis  De Novo Pyrimidine Synthesis Continues from left Formed in cytosol from glutamine GTP from purine synthesis stimulates cytidylate synthetase to balance CTP made from UTP (no purine:pyrimidine branched pathways as in purine synthesis) Ribose 5-phosphate is attached after pyrimidine ring formation CTP impairs de novo Continues to right pyrimidine synthesis to balance UTP:CTP 19  Learning Outcomes Recall the basic structure of nucleotides Describe the de novo synthesis of purine nucleotides and its regulation Describe the de novo synthesis of pyrimdine nucleotides and its regulation Describe how ribonucleotides form deoxyribonucleotides Describe examples relating to nucleotide synthesis and disease 20  Ribonucleotides are the Precursors of Deoxyribonucleotides -OH group in ribonucleotide -H group in deoxyribonucleotide Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides Thioredoxin acts as an intermediate hydrogen-carrying protein that carries a pair of hydrogen atoms, via –SH groups, from NADPH to the ribonucleoside diphosphate Thioredoxin reductase catalyses the reduction of the oxidised form of thioredoxin by NADPH 21  Reduction of Ribonucleotides to Deoxyribonucleotides Reaction catalysed by ribonucleotide reductase (know this enzyme) e e Electrons and H+ are transmitted from NADPH to ribonucleotide reductase via glutaredoxin and thioredoxin e e Glutaredoxin transfers electrons from glutathione (GSH) to ribonucleotide reductase e Thioredoxin transfers pair of H+ to ribonucleotide reductase required to form dNDP Reduced molecules e Oxidised molecules 22  Structure of Ribonucleotide Reductase Activity and substrate specificity of ribonucleotide reductase is regulated by the binding of effector molecules 23 Regulation of Ribonucleotide Reductase by Deoxynucleoside Triphosphates Provides a balanced pool of Regulates overall production precursors for DNA synthesis Next slide Next slide 24  Thymidylate (dTMP) is Derived from dCDP and dUMP DNA contains thymine rather than uracil De novo pathway to thymine only involves deoxyribonucleotides dUTP formed by deamination of dCTP or by phosphorylation of dUDP Next slide Thymidylate synthase catalyses the conversion of dUMP to thymidylate (dTMP) 25 Conversion of dUMP to dTMP is Catalyzed by Thymidylate Synthase Thymidylate synthase catalyzes the conversion of dUMP to dTMP Dihydrofolate reductase reduces dihydrofolate to tetrahydrofolate Folic acid deficiency results in excess incorporation of uracil into DNA leading to DNA damage causing neural tube defects (see slide 28) Derived from folic acid (vitamin B9) 26  Learning Outcomes Recall the basic structure of nucleotides Describe the de novo synthesis of purine nucleotides and its regulation Describe the de novo synthesis of pyrimdine nucleotides and its regulation Describe how ribonucleotides form deoxyribonucleotides Describe examples relating to nucleotide synthesis and disease 27  Folic Acid Deficiency Leads to Reduced Thymidylate Synthesis Folic acid (or vitamin B9) deficiency occurs in ~10% of the human population (and up to 50% of people in impoverished communities) Reduced thymidylate synthesis causes abnormal incorporation of uracil into DNA Repair mechanisms remove the uracil by creating strand breaks that affect the structure and function of DNA – Neural tube defects in infants (during pregnancy) – Associated with cancer, heart disease, and neurological impairment 28 Thymidylate Synthesis and Folate Metabolism as Chemotherapy and Antibiotic Targets 5-Fluorodeoxyuridylate (FdUMP) derived from fluorouracil (salvage pathway) Chemotherapeutic Antibiotic (higher affinity for microbial than mammalian enzyme) Chemotherapeutic and immunosuppressant In aminopterin CH3 = H (methotrexate replaced aminopterin due to synthesis ease) Know dihydrofolate reductase and thymidylate synthase 29  Revision True or false. Ribose 5-phosphate is attached before pyrimidine ring formation. Name the amino acids initially involved in the de novo synthesis of pyrimidines and the end product. True or false. The synthesis of nucleotides in the the de novo synthesis pathway of pyrimidines is linear. Name the enzyme which converts ribonucleotides to deoxyribonucleotides. Name the two functions of this enzyme above in deoxyribonucleotide synthesis. Name the vitamin which gives rise to the substrate that is converted with dUMP to dTMP by thymidylate synthase. True or false. This reaction immediately above is a target of some chemotherapeutic drugs. 30 Summary: De Novo Purine Nucleotide Synthesis Ribose 5-phosphate PRPP Glutamine 5-Phosphoribosylamine 9 steps Glycine (ATPs not shown) Glutamine Formate Aspartate HCO3 IMP Formate Aspartate GTP Adenylosuccinate XMP Glutamine ATP AMP GMP 31 Summary: De Novo Pyrimidine Nucleotide Synthesis Aspartate Carbamoyl phosphate Glutamine Orotate N-Carbamoyl aspartate 2 steps Orotate 2 steps UMP ATP Carbamoyl Aspartate UTP phosphate Glutamine ATP CTP 32