Metabolism MSLSC2001C04 Course Instructor Dr. Gautam Kumar Dr.Gautam Kr. Dept. of Life Sc. 1 Nucleotides Dr.Gautam Kr. Dept. of Life Sc. 2 Biosynthesis and Degradation of Nucleotides Precursors of DNA and RNA: Two types of pathways lead to nucleotides: • The de novo pathways: Synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose 5-phosphate, CO2, and NH3 • de novo pathways for purine and pyrimidine biosynthesis appear to be nearly identical in all living organisms. Origin of the carbon and nitrogen atoms of the purine ring Purine Base Pyrimidine Base • The free bases Guanine, Adenine, Thymine, Cytosine, and Uracil are not intermediates in these pathways 3 Dr.Gautam Kr. Dept. of Life Sc. • Several important precursors are shared by the de-novo pathways for synthesis of Pyrimidines and Purines • Phosphoribosyl pyrophosphate (PRPP) is important in both, and in these pathways • 5-phosphoribosyl-1- pyrophosphate (PRPP) • Amino acid is an important precursor in each type of pathway: Glycine for Purines and Aspartate for Pyrimidines • Aspartate and Glutamine is also used as the source of an amino group in the purine pathways Dr.Gautam Kr. Dept. of Life Sc. 4 De Novo Purine Nucleotide Synthesis Begins with PRPP De novo synthesis of purine nucleotides: Construction of the purine ring of inosinate (IMP) Dr.Gautam Kr. Dept. of Life Sc. 5 Purine ring • The first intermediate with a complete purine ring is inosinate 6 Dr.Gautam Kr. Dept. of Life Sc. (IMP). • The first intermediate with a complete purine ring is inosinate (IMP) Dr.Gautam Kr. Dept. of Life Sc. 7 • Pyrimidine Nucleotides Are Made from Aspartate, PRPP, and Carbamoyl Phosphate The six-membered pyrimidine ring is made first and then attached to Ribose 5-phosphate Required in this process is carbamoyl phosphate, also an intermediate in the urea cycle Carbamoyl phosphate required in pyrimidine biosynthesis is made in the cytosol by a different form of the enzyme, Carbamoyl phosphate synthetase - II Pyrimidine ring is synthesized as Orotate, attached to ribose phosphate, and then converted to the common pyrimidine nucleotides Once Orotate is formed, the ribose 5-phosphate side chain, provided once again by PRPP, is attached to yield orotidylate 8 Dr.Gautam Kr. Dept. of Life Sc. v • Once orotate is formed, the ribose 5- hosphate side chain, provided once again by PRPP, v is attached to yield orotidylate 9 Dr.Gautam Kr. Dept. of Life Sc. • Orotidylate is then decarboxylated to uridylate, which is phosphorylated to UTP. • CTP is formed from UTP by the action of cytidylate synthetase • The nitrogen donor is normally Glutamine, although the cytidylate synthetases in many species can use + NH 4 directly. Dr.Gautam Kr. Dept. of Life Sc. 10 Thymidylate Is Derived from dCDP and dUMP DNA contains thymine rather than uracil De novo pathway to thymine involves only deoxyribonucleotides The immediate precursor of thymidylate (dTMP) is dUMP A one-carbon unit at the hydroxymethyl (-CH2OH) oxidation level is transferred from N5,N10-methylenetetrahydrofolate to dUMP, then reduced to a methyl group Dr.Gautam Kr. Dept. of Life Sc. 11 Ribonucleotides Are the Precursors of Deoxyribonucleotides Reduction of Ribonucleotides to Deoxyribonucleotides by Ribonucleotide reductase • Electrons are transmitted to the enzyme from NADPH by (a) Glutaredoxin or (b) Thioredoxin Ribonucleotide reductase 12 Dr.Gautam Kr. Dept. of Life Sc. Deoxyribonucleoside triphosphate biosynthesis ADP dADP dATP GDP dGDP dGTP CDP dCDP dCTP reductase UDP dUDP dUTP dUMP dTMP Ribonucleotide DTTP Dr.Gautam Kr. Dept. of Life Sc. 13 • Degradation of Purines Produces: Uric Acid T lymphocytes and B lymphocytes do not develop properly Primates excrete much more nitrogen as urea via the Gout is a disease of urea cycle than as uric acid the joints caused by an from purine degradation. elevated concentration of uric acid in the blood and tissues. Similarly, fish excrete much more nitrogen as + NH 4 than as urea produced by the pathway shown here. Dr.Gautam Kr. Dept. of Life Sc. 14 • Degradation of Pyrimidines Produces: Urea The pathways for degradation of pyrimidines generally lead to + NH 4 production and thus to urea synthesis. Thymine, for example, is degraded to methylmalonylsemialdehydean intermediate of valine catabolism. It is further degraded through propionyl-CoA and methylmalonyl-CoA to succinyl- CoA Dr.Gautam Kr. Dept. of Life Sc. 15 Purine and Pyrimidine Bases Are Recycled by Salvage Pathways • The salvage pathways: Recycle the free bases and nucleosides released from nucleic acid breakdown • Free purine and pyrimidine bases are constantly released in cells during the metabolic degradation of nucleotides. The bases are synthesized and then attached to ribose Adenosine phosphoribosyltransferase Free Adinine AMP + PPi Dr.Gautam Kr. Dept. of Life Sc. 16 Lesch-Nyhan syndrome • Children with this genetic disorder, which becomes manifest by the age of 2 years, are sometimes poorly coordinated and mentally retarded. lack of hypoxanthine-guanine phosphoribosyltransferase Many Chemotherapeutic Agents Target Enzymes in the Nucleotide Biosynthetic Pathways • Analogs of glutamine interfere in a number of amino acid and nucleotide biosynthetic pathways • Acivicin shows promise as a cancer chemotherapeutic agent. Dr.Gautam Kr. Dept. of Life Sc. 17 • One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. • Fluorouracil and methotrexate are important chemotherapeutic agents. In cells, fluorouracil is converted to FdUMP, which inhibits thymidylate synthase. • Methotrexate, a structural analog of tetrahydrofolate, inhibits dihydrofolate reductase; the shaded amino and methyl groups replace a carbonyl oxygen and a proton, respectively, in folate Dr.Gautam Kr. Dept. of Life Sc. 18 .