NUCLEOTIDE SYNTHESIS AND DEGRADATION 1. Recognize the structures of the common purine and pyrimidine bases, nucleosides, and nucleotides. 2. Recognize the structure of phosphoribosyl pyrophosphate and phosphoribosylamine. 3. Know the role of tetrahydrofolate derivatives in the synthesis of purine and pyrimidine nucleotides. 4. Describe the salvage pathways by which adenine and guanine are converted back to AMP and GMP. 5. Describe the metabolic pathway for the conversion of purine nucleotides to uric acid. 6. Describe the causes and potential consequences of hyperuricemia. Explain how allopurinol reduces the rate of uric acid formation. 7. Describe the consequences of adenosine deaminase deficiency. 8. Describe how pyrimidine synthesis is affected by the genetic disease orotic aciduria. 9. Describe how deoxyribonucleotides are generated from ribonucleotides. 10. Know the reaction catalyzed by thymidylate synthase. Understand why dihydrofolate reductase is required for this reaction. 11. Explain the actions of methotrexate and fluorouracil as anticancer drugs. NUCLEOTIDE SYNTHESIS AND DEGRADATION 1. Nucleotide structure a. Used for synthesizing DNA and RNA; components of several coenzymes; energy carriers b. Composed of nitrogen-containing base, sugar, and one to three phosphates c. Sugars are ribose or deoxyribose; base is attached to 1’ carbon; molecules with sugar and base known as nucleosides d. Bases commonly found in nucleic acids are adenine and guanine (purines, two rings) and thymine, cytosine, and uracil (pyrimidines, one ring) e. Phosphate is attached to hydoxyl group at 5’ carbon of sugar; nucleosides containing phosphate known as nucleoside (mono-, di- or tri-) phosphates or nucleotides 2. De novo purine nucleotide synthesis a. Begin with ribose 5-phosphate Ring of base constructed through series of reactions that contribute carbon and nitrogen atoms; donated by amino acids (aspartate, glutamine, and glycine), CO2, and N10-formyl-tetrahydrofolate Synthesis of all nucleotides begins with ribose sugar; deoxyribonucleotides are produced by synthesis and subsequent reduction of ribonucleotides b. Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP) Ribose phosphate pyrophosphokinase (PRPP synthetase) uses ATP to add pyrophosphate at 1’ carbon of ribose 5-phosphate PRPP also used for pyrimidine synthesis and salvage pathways PRPP synthetase inhibited by the purine nucleotides AMP, GMP, IMP c. Synthesis of inosine monophosphate (IMP) IMP acts as “parent” purine that can subsequently be converted to AMP or GMP First reaction of pathway is committed step in purine nucleotide synthesis and is regulated - Amide group from glutamine replaces pyrophosphate of PPRP to form 5’phosphoribosylamine - Reaction catalyzed by glutamine:PRPP amidotransferase; enzyme inhibited by the purine nucleotides AMP, GMP, IMP Subsequent steps to get to IMP are not significantly regulated; require energy from four ATP molecules Inhibitors 1 - - Folic acid analogs, such as methotrexate- inhibit reduction of folic acid to tetrahydrofolate (THF) by dihydrofolate reductase; used to treat cancer but toxic to all dividing cells Sulfonamides- inhibit folic acid synthesis in bacteria which is used to make THF; effects specific to bacteria because humans cannot synthesize folic acid and rely on external sources d. Conversion of IMP to AMP and GMP Each conversion occurs via a different two step pathway End product of each pathway (AMP or GMP) inhibits first reaction of that pathway Synthesis of AMP requires GTP for energy, while synthesis of GMP requires ATP; if one nucleotide were low, tend to slow synthesis of the other to restore balance e. Conversion of nucleoside monophosphates (NMPs) to nucleoside di- and triphosphates (NDPs and NTPs) NMPs converted to NDPs by different base-specific kinases (adenylate kinase, guanylate kinase, etc.) NDPs converted to NTPs by nucleoside diphosphate kinase, which has broad specificity ATP is source of transferred phosphate 3. Salvage pathway for purine nucleotide synthesis a. Purines from nucleic acid turnover or external sources can be reconverted to nucleotides b. Base added to PRPP by adenine phosphoribosyl transferase (APRT) to form AMP and by hypoxanthine-guanine phosphoribosyl transferase (HGPRT) to form IMP or GMP c. Nearly complete deficiency of HGPRT causes Lesch-Nyhan syndrome; cannot salvage hypoxanthine and guanine; results in excessive production of uric acid 4. Degradation of purine nucleotides a. Nucleotides converted to nucleosides by phosphatases b. Guanosine degradation Sugar removed from base (guanine) by purine nucleoside phosphorylase Guanine deaminanted to form xanthine, which is oxidized to uric acid by xanthine oxidase c. Adenosine degradation Base on adenosine (adenine) is deaminated by adenosine deaminase to form inosine (hypoxanthine base) Sugar removed from base (hypoxanthine) by purine nucleoside phosphorylase 2 Hypoxanthine oxidized to xanthine and then to uric acid by xanthine oxidase Uric acid cannot be converted to anything else in humans and is excreted in urine d. Gout Hyperuricemia can cause uric acid crystals form in joint fluid leading to arthritic inflammation Contributing factors- certain inherited metabolic disorders, diet rich in meat and seafood, chronic kidney disease, some cancers that cause abnormal cell turnover, chemotherapy treatment of cancers particular certain leukemias and lymphomas that cause rapid cell death Can treat with allopurinol; xanthine oxidase inhibitor; results in excretion of hypoxanthine and xanthine, which are more soluble e. Adenosine deaminase (ADA) deficiency Causes severe combined immunodeficiency (SCID), lack of functional T and B cells Buildup of deoxyadenosine, which leads to buildup of dATP dATP inhibits ribonucleotide reductase (reduction of ribonucleotides to deoxyribonucleotides), thereby interfering with DNA synthesis 5. Pyrimidine synthesis a. Base is made first and then added to sugar; base constructed from amino acids (aspartate, glutamine) and CO2 b. First reaction of pathway is committed step; synthesis of carbamoyl phosphate from glutamine, CO2, and ATP by carbamoyl phosphate synthetase II (CPS II); CPS II inhibited by UTP and activated by ATP and PRPP c. In subsequent steps, aspartate added, ring closure, and an oxidation lead to formation of orotate, which serves as a “parent” pyrimidine base d. Orotate added to PRPP by orotate phosphoribosyl transferase to form the pyrimidine nucleotide orotidine monophosphate (OMP); removal of carboxyl group from OMP by OMP decarboxylase results in UMP; deficiency of orotate phosphoribosyl transferase or decarboxylase results in orotic aciduria, which can be treated with uridine e. CTP is produced by amination of UTP; glutamine supplies nitrogen f. Salvage pathway also exists in which pyrimidines can be reconverted to nucleotides by addition to PRPP; alternatively pyrimidine nucleotides can be cleaved into highly soluble compounds 6. Conversion of ribonucleotides to deoxyribonucleotides a. Reduction of nucleoside diphosphates (ADP, GDP, CDP, UDP) to deoxy forms (dADP, dGDP, dCDP, dUDP); hydrogen atom replaces hydroxyl group at 2’ carbon on ribonucleotides b. Catalyzed by ribonucleotide reductase 3 c. Hydrogen is donated by thioredoxin, a coenzyme component, which becomes oxidized; to regenerate useful thioredoxin, thioredoxin reducatase uses NADPH to reduce thioredoxin d. Ribonucleotide reductase inhibited by all deoxynucleoside triphosphates 7. Synthesis of dTMP from dUMP a. Methylation of dUMP to form dTMP; catalyzed by thymidylate synthase; methyl group contributed by N5, N10-methylene tetrahydrofolate b. Thymidylate synthease inhibited by 5-fluorouracil; used for cancer treatment c. Folic acid analogs such as methotrexate that inhibit dihydrofolate reductase reduce source of donor for methylating dUMP; act to inhibit dTMP synthesis in addition to purine synthesis 4