Nucleotide Synthesis and Metabolism 1 Lecture Notes PDF

Dr Icolyn Amarakoon Email: [email protected] OFFICE LOCATION: LEVEL 2 Block A- Offices behind the Molecular Biology lab {through the glass doors and it’s the 1st door on the right}  LEARNING OUTCOMES At the end of the metabolism lectures you should be able to…… Describe how...

Dr Icolyn Amarakoon Email: [email protected] OFFICE LOCATION: LEVEL 2 Block A- Offices behind the Molecular Biology lab {through the glass doors and it’s the 1st door on the right}  LEARNING OUTCOMES At the end of the metabolism lectures you should be able to…… Describe how nucleotides are synthesised in the cell Differentiate between the 2 pathways for nucleotide biosynthesis De novo and salvage pathways Describe the de novo and salvage synthesis of purines Synthesis of IMP (precursor of adenine and guanine) synthesis of adenine and guanine from IMP Describe the de novo and salvage synthesis of pyrimidines Synthesis of uracil, cytosine and thymine Discuss the degradative processes of purines and pyrimidines Explain the synthesis of deoxyribonucleotides from ribonucleotides  NUCLEIC ACID METABOLISM Nucleic Acid metabolism involves the processes by which nucleotides are biosynthesized and broken down. Biosynthesis reactions are anabolic, and usually involves reactions of phosphate, pentose sugar, and nitrogenous base. Breakdown reactions are catabolic  TWO PATHWAYS TO NUCLEOTIDE SYNTHESIS Salvage pathway recycles the free De novo pathway begins with bases and nucleosides released from metabolic precursors: Amino acids, nucleic acid breakdown ribose 5-phosphate, CO2 and NH3  NUCLEIC ACID METABOLISM OVERVIEW Metabolic requirements for the nucleotides and their cognate bases met by both dietary intake or synthesis de-novo (new synthesis from scratch) from low molecular weight precursors The salvage pathways (reuse those we already have) are a major source of nucleotides for synthesis of DNA, RNA and enzyme co- factors. NOTE : Nucleotides are synthesized FIRST as ribonucleotides (whether by de novo or salvage) and then converted to deoxyribonucleotides as needed to make DNA.  NUCLEIC ACID METABOLISM OVERVIEW Both the Salvage and De novo synthesis pathways of purine and pyrimidine biosynthesis lead to production of nucleoside-5'-phosphates through the utilization of : an activated sugar intermediate 5-phosphoribosyl-1- pyrophosphate (PRPP) PRPP is generated by the action of PRPP synthetase and requires ATP and a class of enzymes called Phosphoribosyltransferases. BIOSYNTHESIS OF PURINE RIBONUCLEOTIDES Nucleic Acid Metabolism  DE NOVO SYNTHESIS OF PURINES occurs mainly in the liver Synthesis of the purine nucleotides begins with 5- phosphoribosyl-1-pyrophosphate (PRPP) and leads to the first fully formed nucleotide, Inosine 5'- MonoPhosphate (IMP)  STEP 1: PRPP PRODUCTION STEP 1 (regulated step): conversion of ribose-5-phosphate to phosphoribose-1- pyrophosphate (PRPP) PRPP is generated by the action of PRPP synthetase and ATP The reaction releases AMP + 2 high energy phosphate consumed  STEP 2: SYNTHESIS OF INOSINE MONOPHOSPHATE (IMP) The rate limiting, committed step is catalyzed by glutamine- PRPP amidotransferase to form 5-phosphoribosyl amine SYNTHESIS OF IMP  STEP 3: SYNTHESIS OF AMP AND GMP FROM IMP IMP represents a branch point for purine biosynthesis, because it can be converted into either AMP or GMP through two distinct reaction pathways. STEP 3: SYNTHESIS OF AMP AND GMP FROM IMP NOTE The pathway leading to AMP requires energy in the form of GTP; that leading to GMP requires energy in the form of ATP. The utilization of GTP in the pathway to AMP synthesis allows the cell to control the proportions of AMP and GMP to near equivalence. The accumulation of excess GTP will lead to accelerated AMP synthesis from IMP instead, at the expense of GMP synthesis. DE NOVO SYNTHESIS OF PURINES Note in de novo purine biosynthesis the base is "built" on the ribose sugar. Note the intermediates involved in synthesis of the purine ring  REGULATION OF PURINE NUCLEOTIDE SYNTHESIS The essential rate limiting steps in purine biosynthesis occur at the first two steps of the pathway. The synthesis of PRPP by PRPP synthetase is feed-back inhibited by purine-5'-nucleotides (AMP and GMP) The amidotransferase reaction catalyzed by glutamine-PRPP amidotransferase is also feed-back inhibited by ATP, ADP and AMP at one inhibitory site and GTP, GDP and GMP at another. Conversely the activity of the enzyme is stimulated by PRPP. REGULATION OF PURINE NUCLEOTIDE SYNTHESIS  Synthetic Inhibitors Sulfonamides e.g. Amoxicillin, Bactrim, Gantanol – (inhibit the growth of rapidly dividing microorganism without interfering with the cell function) Structural analogs of folic acid e.g. Methotrexate: (control spread of cancer by interfering in the synthesis of nucleotide) SUMMARY: PURINE DE NOVO SYNTHESIS PRPP is the source of the sugar for purine nucleotides. It is synthesized from ribose 5-phosphate of the pentose phosphate pathway. All purine N2 come from amino acids (glutamine, aspartate & glycine). The branch point in purine biosynthesis is inosine 5'- monophosphate (IMP) The conversion of IMP to either AMP or GMP is also highly regulated - AMP feedback inhibits the first step from IMP to AMP and GMP feedback inhibits the first step from IMP to GMP. ATP is required to synthesize GMP and GTP is required to make AMP  SUMMARY OF MAIN CONCEPTS IMP is synthesized through the assembly of a purine base on ribose-5-phosphate.  Kinases convert IMP-derived AMP and GMP to ATP and GTP.  Purine nucleotide synthesis is regulated by feedback inhibition & feed forward activation. SALVAGE PATHWAY: SYNTHESIS OF NUCLEOTIDES Salvage pathway: A recycling metabolic pathway in which biomolecules such as nucleotides are synthesized from intermediates-which would otherwise be waste products-in the degradative pathway. Salvage of free nucleotides: consumes much less energy than de novo nucleotide synthesis is the energetically preferred source of nucleotides for nucleic acid synthesis SALVAGE PATHWAY: SYNTHESIS OF PURINE NUCLEOTIDES Salvage pathway occurs primarily in extrahepatic tissues in which de novo synthesis can not take place eg. brain and RBCs, whereas de novo pathway occurs primarily in the liver. The free purine bases, adenine, guanine, and hypoxanthine, can be reconverted to their corresponding nucleotides by phosphoribosylation. Salvage accounts for 90% of daily purine nucleotide biosynthesis.  SALVAGE PATHWAY: SYNTHESIS OF PURINE NUCLEOTIDES Most purine bases are recycled rather than degraded. Salvage pathways require less energy than the de novo pathway for purine synthesis. Two enzymes (APRT, HGPRT) are involved in salvage of purine nucleotides. adenosine phosphoribosyl transferase (APRT) hypoxanthine-guanine phosphoribosyl transferase (HGPRT) is responsible for most of the recycling.  SALVAGE PATHWAY Two key transferase enzymes are involved in the salvage of purines: adenosine phosphoribosyltransferase (APRT), which catalyzes the following rxn: Adenine + PRPP AMP + Ppi hypoxanthine-guanine phosphoribosyl transferase (HGPRT), which catalyzes the following rxns: Hypoxanthine + PRPP IMP + Ppi Guanine + PRPP GMP + PPi SALVAGE PATHWAY- ADENINE Adenosine Phosphoribosyl Transferase (APRT)  SALVAGE PATHWAY: HYPOXANTHINE & GUANINE  HGPRT use PRPP as the source of ribose-5- P  HGPRT catalyses the transfer of ribose-5-P into hypoxanthine or guanine to regenerate IMP or GMP SALVAGE AND DE NOVO PATHWAYS SALVAGE PATHWAY: SYNTHESIS OF NUCLEOTIDES If purines are not salvaged, glutamine-PRPP amidotransferase in the de novo purine biosynthetic pathway will be activated to provide missing nucleotides: More PRPP will be available to stimulate glutamine-PRPP amidotransferase. There will be a decrease in the nucleotides (AMP, IMP, GMP) available to inhibit de novo synthesis. CATABOLISM OF PURINE NUCLEOTIDES Nucleic Acid Metabolism CATABOLISM OF PURINE NUCLEOTIDES Catabolism produces purine bases for salvage pathways Catabolism of the purine nucleotides leads ultimately to the production of uric acid which is insoluble and is excreted in the urine as sodium urate crystals  First step in adenosine degradation is deamination First step in guanosine degradation is de-ribosylation Uric acid build up can cause problems in some individuals.  Catabolism of Purine Nucleotides A critically important enzyme of purine salvage in rapidly dividing cells is adenosine deaminase (ADA) which catalyzes the deamination of adenosine to inosine. Deficiency in ADA results in the disorder called severe combined immunodeficiency (SCID)  SEVERE COMBINED IMMUNODEFICIENCY (SCID) Fatal in children (die by age 2) if untreated. Treatment: Bone marrow replacement therapy (human leukocyte antigen HLA) Enzyme replacement therapy- (synthetic ADA) DISORDERS OF PURINE METABOLISM Immunodeficiency diseases: enzyme defects are adenosine deaminase (ADA)- deficiency causes severe combined immunodeficiency (SCID)- {T-cell and B-cell dysfunction, apoptosis}. purine nucleoside phosphorylase- deficiency is associated with impairment of T-cell function but has no effect on B- cell function. Uric acid synthesis is decreased and the tissue levels of purine nucleosides and nucleotides are higher. DISORDERS OF PURINE METABOLISM HYPERURICEMIA AND GOUT Uric acid is the end product of purine metabolism in humans. The normal concentration of uric acid in the serum of adults is in the range of 3-7 mg / dl. In women, it is slightly lower ( by about 1 mg ) than in men. The daily excretion of uric acid is about 500-700 mg.  HYPERURICEMIA AND GOUT Hyperuricemia refers to an elevation in the serum uric acid concentration. This is sometimes associated with increased uric acid excretion (Uricosuria) GOUT is metabolic disease associated with overproduction of uric acid. At the physiological pH, uric acid is found in a more soluble form as sodium urate. In severe hyperuricemia, crystals of sodium urate get deposited in the soft tissues, particularly in the joints. Uric acid stones in kidney (urolithiasis)  CHRONIC GOUT TREATMENT Allopurinol is a structural analog of hypoxanthine. It inhibits uric acid synthesis in patients overproducing uric acid DISORDERS OF PURINE METABOLISM PRPP glutamylamidotransferase: lack of feedback control of this enzyme by purine nucleotides also leads to their elevated synthesis. HGPRT deficiency: This is an enzyme of purine salvage pathway, its defect causes Lesch- Nyhan syndrome. increased synthesis of purine nucleotides by a two fold mechanism: Firstly, decreased utilization of purines ( Hypoxanthine & guanine ) by salvage pathway, resulting in the accumulation & diversion of PRPP for purine nucleotides. Secondly, the defect in salvage pathway leads to decreased levels of IMP & GMP causing impairment in the tightly controlled feedback regulation of their production. Disorders of Nucleotide Metabolism NATURE OF DISORDER DEFECT COMMENTS DEFECT Gout 3 enzyme defects can lead to gout: Hyperuricemia, * PRPP synthetase activity up disease of the joint *HGPRT deficiency Lesch-Nyhan syndrome HGPRT lack of enzyme- Seen in male defect in gene children. Intellectual deficits Severe Combined adenosine deaminase lack of enzyme Lack effective Immunodeficiency (SCID) (ADA) immune system Renal lithiasis (kidney APRT lack of enzyme rare type of urinary stones) stone disease Xanthinuria Xanthine oxidase lack of enzyme hypouricemia and xanthine renal lithiasis  REGULATION OF NUCLEOTIDE BIOSYNTHESIS: PURINES Synthesis of PRPP and conversion of PRPP to phosphoribosylamine are BOTH inhibited by the purine nucleotides IMP, AMP and GMP. (Important enzyme is glutamine phosphoribosyl amidotransferase, which catalyzes PRPP to phosphoribosylamine) Conversion of IMP to AMP is inhibited by AMP and conversion of IMP to GMP is inhibited by GMP; and AMP synthesis requires GTP energy while GMP synthesis requires AMP energy.  DRUGS THAT AFFECT NUCLEOTIDE BIOSYNTHESIS Critical in cancer treatment. Chemotherapy: Inhibit specific enzymes of nucleotide metabolism. Ultimately prevent cell or virus proliferation by inhibiting RNA or DNA synthesis Hydroxyurea, anti-folates, FdUMP (Fluorodeoxyuridylate) 5-Fluorouracil (5-FU), Methotrexate (anti-inflammatory, immunosuppressive) Antiviral therapies Acyclovir and herpes Azidothymidine -AZT (Zidovudine, nucleotide analogue)