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Nucleotide metabolism 1 1-Biochemistry Lippincott’s Illustrated Reviews Sixth Edition chapter 22 2- MARKS’ Basic Medical Biochemistry A Clinical Approach Chapter 41  Outline Nucleotide synthesis. A- Purine synthesis. Purine salvage pathway....

Nucleotide metabolism 1 1-Biochemistry Lippincott’s Illustrated Reviews Sixth Edition chapter 22 2- MARKS’ Basic Medical Biochemistry A Clinical Approach Chapter 41  Outline Nucleotide synthesis. A- Purine synthesis. Purine salvage pathway. Purines degradation. B- Pyrimidine synthesis. Salvage of Pyrimidine Pyrimidines degradation. Synthesis of deoxyribonucleotides that are necessary for DNA synthesis. Thymine nucleotide synthesis.  INTRODUCTION § Nucleotides are building blocks of nucleic acids ( DNA & RNA). § They are non-essential nutrients , because they can be synthesized in the body. § Nucleotides enter the structure of ATP, the main source of energy in the cell. § Nucleotides enter the structure of many coenzymes as NAD, NADP and FAD. § Nucleotides enter in the structure of cAMP and cGMP which act as secondary messenger of many hormones. § Nucleotides are important regulatory compounds for many of the pathways of intermediary metabolism, inhibiting or activating key enzymes. § The purine and pyrimidine bases found in nucleotides can be synthesized de novo, or can be obtained through salvage pathways.. § Nucleic acids are linear polymers, specialized for the storage and transmission of genetic information for cellular growth and reproduction § Purine base contains adenine and guanine § Pyrimidine base contains cytosine, uracil and thymine. Purine and pyrimidine structures: § T and U differ in that only T has a methyl group.  Unusual bases Acetylation Some species have unusual (modified) bases in their DNA and RNA for example: – some viral DNA – tRNA Base modifications include: Reduction – Methylation – Acetylation – Reduction. Methylation – Glycosylation  Nucleosides Addition of a pentose sugar (ribose/deoxyribose) to a base (purine/pyrimidine) produces a nucleoside through an N-glycosidic linkage – Ribose è ribonucleoside – 2-Deoxyribose è deoxyribonucleoside Base DNA RNA Adenine deoxyadenosine adenosine Guanine deoxyguanosine guanosine Cytosine deoxycytidine cytidine Thymine deoxythymidine - Uracil - Uridine  Nucleotides § Addition of one or more phosphate groups to a nucleoside produce a nucleotide. § Nucleotide consists base (purine – pyrimidine), pentose (ribose- deoxyribose) and phosphates § Phosphate bind C5 atoms of the pentose sugar through ester linkage to the 5'-OH of the pentose.  Nucleotides Two Important Points 1. The phosphate groups are responsible for the net negative charge associated with DNA and RNA. One phosphate group is attached to pentose -> nucleoside monophosphate (e.g adenosine monophosphate (AMP, adenylate). Second or third phosphate is added to pentose -> a nucleoside diphosphate (e.g adenosine diphosphate (ADP) or triphosphate (e.g adenosine triphosphate ( ATP). Naming nucleotides  There are 3 pathways leading to nucleotides 1. De novo synthesis: The synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO2, and one-carbon units. All the enzymes involved in the purine nucleotide synthesis and desecration are found in the in cytosol of liver, small intestine and thymus. 2. Salvage pathways: The synthesis of nucleotide by recycle the free bases or nucleosides released from nucleic acid breakdown. Salvage pathways are used to recover bases and nucleosides that are formed during degradation of RNA and DNA 3. Conversion of ribonucleotide to deoxyribonucletoides The conversion of ribonucleotides to deoxyribonucleotides occurs at the level of the nucleoside diphosphates. All four ribonucleotides—ADP, CDP, GDP and UDP—are reduced by the same enzyme, ribonucleotide reductase (RR)  Purine Biosynthesis § Purine ring atoms are contributed by a number of compounds. § The purine ring is synthesis by a series of reaction that add the donated carbons and nitrogens to a performed ribose 5-phosphate. § In humans, all necessary enzymes are found in the cytoplasm of the cell. Most de novo synthesis occurs in the liver.  Step1: 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis Pentose phosphate pathway Glucose End product inhibition (PRPP:Low) Salvage This step involve the production of 5-phosphoribosyl-1-pyrophosphate pathway (PRPP) from ribose-5-phosphate with ATP by the enzyme phosphoribosyl (PRPP: high) phosphate synthetase (PRPP synthetase). De novo Although this enzyme is regulated but it is not the committed step in pathway purine synthesis pathway.  Step 2: Synthesis of 5’-phosphoribosylamine Activator : PRPP Glutamine phosphoribosyl Inhibitors: GMP, pyrophosphate AMP and the end amidotransferase (GPAT) product of the pathway Highly regulated enzyme (rate limiting step) this the committed step in purine nucleotide biosynthesis Step 2: Synthesis of 5’-phosphoribosylamine glutamine provide N9 of the purine ring Step 3: Synthesis of glycinamide ribosyl 5- phosphate or glycinamide ribotide The entire glycine molecule is added to the growing precursor. Glycine provide C4, C5 and N7 of the purine ring. This step require energy in the form of ATP.  Glycinamide ribosyl 5- phosphate Step 4 N-formyl FH4 (C8) Glutamine (N3)+ ATP+ATP to Step 5, 6 close the ring Step 7 CO2 (C6) Aspartate (N1) +ATP Step 8, 9 Step 10 N-formyl FH4 (C2) Step 11 ring closure to form IMP Inosine monophosphate (IMP) In order to form IMP 6 ATP is needed from the beginning of Ribose 5-P  Adenosine and guanosine monophosphates synthesis Inosine monophosphate (IMP) NAD GTP + + _ _ + IMP dehydrogenase +H‫ـ‬2O Aspartate Adenylosuccinate synthetase (oxidation) NADH GDP+ Pi XMP Adenylosuccinate (xanthosine monophosphate) ATP + Adenylosuccinase GMP Glutamine synthetase Fumarate AMP+PPi Glutamate AMP GMP (Adenosine monophosphate) (Guanosine monophosphate) Cross regulation ADP GDP ATP GTP  oxidation of C2 of purine 4 Carbon removed  Conversion of nucleoside monophosphates to nucleoside diphosphates and triphosphates q NDP are synthesis from the NMP by base specific nucleoside monophosphate kinases q This kinases do not discriminate between ribose or deoxyribose in the substrate q ATP is the source of the transferred phosphate because it is present in higher concentration than other nucleoside triphosphate q Adenylate kinase is active in liver, muscle where the turnover of energy from ATP is high q It is function to maintain an equilibrium among AMP, ADP and ATP q NDP and NTP are interconverted by nucleoside diphosphate kinase Synthetic inhibitors of purine synthesis Sulfonamides inhibit the growth of microorganism without interfering with human cell functions. Analogs of PABA (para-aminobenzoic acid) inhibit bacterial synthesis of folic acid (TH4 coenzyme) Methotrexate: Structural analogue of folic acid. It control the spread of cancer by interfering with nucleotide synthesis -> DNA, RNA synthesis  Methotrexate and Cancer Dihydropteroate Dihydrofolate synthase reductase Methotrexate (chemotherapy) : Affects rapidly growing cells by inhibiting the enzyme that convert Folic acid to tetrahydrofolate (dihydrofolate reductase). Toxic to all dividing cell. Nucleotide metabolism 2 1-Biochemistry Lippincott’s Illustrated Reviews Sixth Edition chapter 22 2- MARKS’ Basic Medical Biochemistry A Clinical Approach Chapter 41  Purine salvage pathway t Die DNA and RNA Nucleoside or degradation free bases pathway Salvage Nucleotides for RNA and DNA synthesis Enzyme de novo synthesis of purine are deficient in some tissue such as brain cell, red cells and white blood cells.  Purine salvage pathway Two enzymes are involved: Ø HGPRT Ø APRT Both enzymes use PRPP as a source of ribose 5-phosphate Irreversible because of the release of pyrophosphate by pyrophosphatase. Absence of HGPRT is the cause of Lesch-Nyhan syndrome. Lesch-Nyhan syndrome: § is a rare inherited disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT)>> inability to salvage hypoxanthine or guanine which causes a accumulation of uric acid in the body: § Lack of salvage pathway >> increases PRPP levels and decreases IMP and GMP levels. § Increases PRPP levels >> De novo pathway increases. § Decrease purine reutilization + Increase purine synthesis >> increase purine degradation >> increase uric acid. § Excess uric acid can be released from the blood and build up in soft tissue or joints (causing gouty arthritis) and can also cause kidney stones. § It is characterized by behavioral disturbances including self-mutilation (biting of lips and fingers). Synthesis of deoxyribonucleotides The nucleotide required for DNA synthesis are 2’-deoxyribonucleotides which are produced from ribonucleoside diphosphates by Ribonucleotide reductase enzyme – Specific for reduction of purine/pyrimidine nucleoside diphosphates (NDP) to their deoxy form (i.e. ADP===dADP) – The immediate donors of hydrogen atoms are two SH groups on the enzyme itself – Reduced enzyme regeneration (thioredoxin as coenzyme) – Reduced thioredoxin regeneration (NADPH + H+) Regulation of deoxyribonucleotide synthesis Ribonucleotide reductase is responsible for maintaining a supply of the deoxyribonucleotides required for DNA synthesis The regulation of the enzyme is complex, in addition to catalytical site, R1 contains two allosteric sites involved in regulating enzymatic activity 1) Activity sites: binding dATP inhibit enzyme activity while binding ATP activate the enzyme 2) Substrate specificity sites: the binding of different NTP (ATP, dATP, dTTP or dGTP) to substrate specificity sites on the enzyme regulate the substrate specificity>>increase the conversion of ribonucleotides to deoxyribonucleotides  Purine salvage pathway t Die DNA and RNA Nucleoside or degradation free bases pathway Salvage Nucleotides for RNA and DNA synthesis Enzyme de novo synthesis of purine are deficient in some tissue such as brain cell, red cells and white blood cells. Degradation of purine nucleotides Degradation of dietary nucleic acids: Occur in small intestine RNA and DNA released from food in the intestinal tract are degraded to oligonucleotides by pancreatic ribonucleases and deoxyribonucleases Oligonucleotide are hydrolysed by pancreatic phosphdiesterase to mononucleotides Mononucleotides are hydrolyzed to nucleosides by nucleotidases (remove P) Nucleosides are degraded by nucleosidases to free purine and pyrimidines bases + (deoxy)ribose-1- phosphate. Inside the cells, purine bases are oxidized to uric acid and pyrimidines bases are oxidized to CO2 and ammonia which are excreted in the urine  Formation of uric acid 1. An amino group is removed from AMP to produce IMP or from adenosine to produce inosine by AMP deaminase or adenosine deaminase 2. IMP and GMP are converted into their nucleoside forms inosine and guanosine by the action of 5’ nucleotidase 3. Inosine and guanosine convert into their purine bases by purine nucleoside phosphorylase  Formation of uric acid 4. Guanine is deaminated to form xanthine by guanase 5. Hypoxanthine is oxidized by xanthine oxidase (XO) to xanthine which oxidized to uric acid by xanthine oxidase Uric acid is the final product of purine degradation and is excreted in the urine Diseases associated with purine degradation 1- Gout Definition: Gout is a disorder that initiated by high levels of uric acid in blood (hyperuricemia) either by overproduction or underexcretion of uric acid. Hyperuricemia leads to deposition of monosodium urate (MSU) crystals in joints and soft tissues or forms uric acid stones in kidney.  Diagnosis § Definitive diagnosis only possible by aspirating and inspecting fluid from infected joints § Polarized microscopy confirm the presence of needle-shaped MSU crystals  Treatment of gout A. Acute attack: anti- inflammatory drugs B. Decrease uric acid synthesis (for example: allopurinol inhibits XO enzyme) Classification of Hyperuricemia A- Uric acid underexcretion – Accounts for >90% of hyperuricemia – Unidentified Inherited excretory or disease that affects renal excretion of uric acid – Environmental factors: drugs or exposure to lead. B- Uric acid overproduction – Accounts for 10% of hyperuricemia – Genetic disorders: (gene mutation) a) Over activity of PRPP synthetase >> increases PRPP>> increases purine production >> increases uric acid in blood b) Lesch-Nyhan syndrome >> caused by decreased salvage of hypoxanthine and guanine >>Deficiency HGPRT >> increases uric acid in blood – High rate of cell turnover >> increases purine degradation such as in patients with myeloproliferative disorders or under chemotherapy or as a result of unrelated metabolic diseases.  2- Adenosine deaminase (ADA) deficiency ADA enzyme is expressed in all cells. Human lymphocytes (immune cells) have the highest activity of this cytoplasmic enzyme A deficiency of ADA results in (accumulation of adenosine>> converts to ATP and dATP (dATP accumulation) >> Ribonucleotidase reductase is inhibited by dATP >> preventing the production of all deoxyribose containing nucleotide >> can not make DNA and divide – It causes a severe combined immunodeficiency disease (SCID) decreasing both B and T lymphocytes. – Treatment requires either bone marrow transplantation, enzyme replacement therapy and gene therapy – Without treatment : children die at age of 2 years. Nucleotide metabolism 3 1-Biochemistry Lippincott’s Illustrated Reviews Sixth Edition chapter 22 2- MARKS’ Basic Medical Biochemistry A Clinical Approach Chapter 41 § Outline: § Synthesis of pyrimidine (De novo, Salvage) § Degradation of pyrimidine  Pyrimidine synthesis A- sources of pyrimidine atoms: Unlike purine synthesis, The base is synthesized first then attached to the ribose 5-phosphate sugar  First step in the De Novo pyrimidine synthesis pathway Carbamoyl phosphate synthesis : CO2 - UTP (end product) + PRPP This reaction is analogous to which reaction???? Pyrmidine synthesis Differences between CPSI and CPSII  Regulation of the de novo pyrimidine synthesis CPS II enzyme is inhibited by UTP and activated by PRPP. The activity is also regulated by the cell cycle. At the end of the S phase the inhibition by UTP is more pronounced and the activation by PRPP is reduced As the cells approach S-phase CPS II become more sensitive to PRPP activation and less sensitive to UTP inhibition  De Novo pyrimidine synthesis pathway Carbamoyl phosphate synthesis Orotic acid synthesis Pyrimidine nucleotide UDP synthesis UTP Pyrimidine nucleotide synthesis Cytidine triphosphate (CTP) synthesis – CTP is produced by amination of UTP by CTP synthetase Amination CDP dCDP dCTP Pyrimidine nucleotide synthesis Deoxythymidine monophosphate synthesis (dTMP) Deamination dUMP is converted to dTMP by thymidylate synthase Inhibitor of thymidylate synthase enzyme is (5- flurouracil) Inhibitor of Dihydrofolate reductase is (Methotrexate) These drugs decrease supply of THF which causes: 1. Inhibition of purine synthesis 2. Prevention methylation of dUMP to dTMP lower cellular conc. of essential comp. of DNA → lower DNA synthesis → slower cell growth → drugs are used to decrease the growth rate of cancer cells dTDP dTTP  Salvage of pyrimidine bases Pyrimidine bases are normally salvaged by a two steps route: 1- Non specific pyrimidine nucleoside phosphorylase which convert pyrimidine base to their respective nucleosides Uridine or phosphorylase Thymidine phosphorylase  Salvage of pyrimidine bases 2- The more specific nucleoside kinase then reacts with the nucleoside to form nucleotide Enzyme Reaction Uridine + ATP UMP +ADP Uridine-cytidine kinase Cytidine + ATP CMP +ADP Deoxythymidine kinase dthymidine + ATP dTMP +ADP Deoxycytidine kinase dcytidine +ATP dCMP +ADP The nucleoside phosphorylase – Nucleoside kinase route for the synthesis of pyrimidine nucleoside monophosphate is relatively inefficient for salvage of pyrimidine bases because of the very low concentration of the base in plasma and tissues. Degradation of pyrimidine Pyrimidine nucleotide dephosphorylated Pyrimidine nucleoside cleaved Free Pyrimidine + ribose 1 phosphate  Degradation of pyrimidine CO2 cytosine deaminated uracil + NH4+ + β- alanine CO2 thymine + NH4+ + β- aminoisobutyrate urine Good Luck