Nucleotide metabolism Hamad Yaseen, PhD MLS Department, FAHS, KU [email protected] Outline • Review: Structure of nucleic acid • Degradation of nucleic acid • Synthesis of Purine Nucleotides • Degradation of Purine Nucleotides • Synthesis of Pyrimidine Nucleotides • Degradation of Pyrimidine Nucleotides Nucleoside and Nucleotide! Nucleoside = Nitrogenous base ribose Nucleotide = Nitrogenous base ribose phosphate Purines vs Pyrimidines Structure of nucleotides! pyrimidine! OR! purine! N-β-glycosyl! bond! Ribose! or! 2-deoxyribose! Section 1 Degradation of nucleic acid Degradation of nucleic acid Nucleoprotein In stomach Gastric acid and pepsin Nucleic acid Protein In small intestine Endonucleases: RNase and DNase Nucleotide Nucleotidase Phosphate Nucleoside Nucleosidase Base Ribose Significances of nucleotides 1. Precursors for DNA and RNA synthesis 2. Essential carriers of chemical energy, especially ATP 3. Components of the cofactors NAD+, FAD, and coenzyme A 4. Formation of activated intermediates such as UDP-glucose and CDP-diacylglycerol. 5. cAMP and cGMP, are also cellular second messengers. Section 2 Synthesis of Purine Nucleotides There are two pathways leading to nucleotides • De novo synthesis: The synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO2, and one-carbon units. • Salvage pathways: The synthesis of nucleotide by recycle the free bases or nucleosides released from nucleic acid breakdown. De novo synthesis • Site: – in cytosol of liver, small intestine and thymus • Characteristics: a. Purines are synthesized using 5- phosphoribose(R-5-P) as the starting material step by step. b. PRPP(5-phosphoribosyl-1-pyrophosphate) is active donor of R-5-P. c. AMP and GMP are synthesized further at the base of IMP(Inosine-5'-Monophosphate). Purines Structure Element sources of purine bases 6 5 7 1 8 N10-Formyltetrahydrofolate 2 4 9 3 N10-Formyltetrahydrofolate First, synthesis Inosine-5'-Monophosphate, IMP Synthesis of Inosine Monophosphate (IMP) • Basic pathway for biosynthesis of purine ribonucleotides • Starts from ribose-5-phosphate(R-5-P) • Requires 11 steps overall • occurs primarily in the liver • How many ATP molecules are used?? The big picture OH Step 1:Activation of ribose-5-phosphate R5P Committed step ATP 1 ribose phosphate pyrophosphokinase AMP Step 2: acquisition of purine atom N9 2 Gln:PRPP amidotransferase • Steps 1 and 2 are tightly regulated by feedback inhibition Step 3: acquisition of purine atoms C4, C5, and N7 3 glycinamide synthetase • Step 4: acquisition of purine atom C8 4 GAR transformylase Step 5: acquisition of purine atom N3 5 • Step 6: closing of the imidazole ring 6 Step 7: acquisition of C6 7 AIR carboxylase Carboxyaminoimidazole ribonucleotide (CAIR) Step 8: acquisition of N1 Carboxyaminoimidazole ribonucleotide (CAIR) SAICAR synthetase Step 9: elimination of fumarate adenylosuccinate lyase Step 10: acquisition of C2 AICAR transformylase Step 11: ring closure to form IMP • Once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells). The big picture In Class Activity *** Calculate how many ATP equivalents are needed for the de novo synthesize IMP. Assume that all of the substrates (R5P, glutamine, etc) are available Note: You should be able to do this calculation for the synthesis of any of the nucleoside monophosphates Summary • The purine ring is built on a ribose-5-P foundation • Step 1: ribose-5-P must be activated - by PPi – PRPP is limiting substance for purine synthesis – But PRPP is a branch point so next step is the committed step – • Step 2: Gln PRPP amidotransferase – Note that second step changes C-1 configuration – G- and A-nucleotides inhibit this step - but at distinct sites! • Step 3: Glycine carboxyl condenses with amine – Glycine carboxyl activated by -P from ATP – Amine attacks glycine carboxyl • Step 4: Formyl group of N10-formyl-THF is transferred to free amino group of GAR • Step 5: C-4 carbonyl forms a P-ester from ATP and active NH3 attacks C-4 to form imine Closure of the first ring, carboxylation and attack by aspartate • Step 6: Similar in some ways to step 5. ATP activates the formyl group by phosphorylation, facilitating attack by N. • Step 7: Carboxylation probably involves electron "push" from the amino group • Step 8: Attack by the amino group of aspartate links this amino acid with the carboxyl group Loss of fumarate, another 1-C unit and the second ring closure • Step 9: Deprotonation of Asp-CH2 leads to cleavage to form fumarate • Step 10: Another 1-C addition catalyzed by THF • Step 11: Amino group attacks formyl group to close the second ring Note that 5 steps use ATP, but that this is really six ATP equivalents! Dependence on THF in two steps means that methotrexate and sulfonamides block purine synthesis 3. Conversion of IMP to AMP and GMP Note: GTP is used for AMP synthesis. Note: ATP is used for GMP synthesis. IMP is the precursor for both AMP and GMP. Making AMP and GMP Reciprocal control occurs in two ways • GTP is the energy input for AMP synthesis, whereas ATP is energy input for GMP • AMP is made by N addition from aspartate (in the familiar way) • GMP is made by oxidation at C-2, followed by replacement of the O by N (from Gln) • Last step of GMP synthesis is identical to the first two steps of IMP synthesis Regulation of de novo synthesis The significance of regulation: (1) Meet the need of the body, without wasting. (2) AMP and GMP control their respective synthesis from IMP by a feedback mechanism, [GTP]=[ATP] • Purine nucleotide biosynthesis is regulated by feedback inhibition Salvage pathway • Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis can be directly converted to the corresponding nucleotides. • The significance of salvage pathway : – Save the fuel. – Some tissues and organs such as brain and bone marrow are only capable of synthesizing nucleotides by salvage pathway. • Two phosphoribosyl transferases are involved: – APRT (adenine phosphoribosyl transferase) for adenine. – HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine. Purine Salvage and Lesch-Nyhan syndrome • Salvage pathways collect hypoxanthine and guanine and recombine them with PRPP to form nucleotides in the HGPRT reaction • Absence of HGPRT is cause of Lesch-Nyhan syndrome • In L-N, purine synthesis is increased 200-fold and uric acid is elevated in blood • This increase may be due to PRPP feed-forward activation of de novo pathways Purine Salvage Pathway . adenine phosphoribosyl transferase Adenine AMP PRPP PPi O O N N N N 2-O POH C N N 3 2 O N hypoxanthine-guanine N phosphoribosyl transferase Hypoxanthine (HGPRT) HO OH IMP O O PRPP N PPi N N N N N NH 2-O POH C N N NH 2 3 2 O 2 Guanine HO OH GMP . Absence of activity of HGPRT leads to Lesch-Nyhan syndrome. Lesch-Nyhan syndrome • first described in 1964 by Michael Lesch and William L. Nyhan. • there is a defect or lack in the HGPRT enzyme • Sex-linked metabolic disorder: only males • the rate of purine synthesis is increased about 200-fold – Loss of HGPRT leads to elevated PRPP levels and stimulation of de novo purine synthesis. • uric acid level rises and there is gout • in addition there are mental aberrations • patients will self-mutilate by biting lips and fingers off Lesch-Nyhan syndrome Degradation of Purine Nucleotides Purine Degradation Purine catabolism leads to uric acid • Nucleotidases and nucleosidases release ribose and phosphates and leave free bases • Xanthine oxidase and guanine deaminase route everything to xanthine • Xanthine oxidase converts xanthine to uric acid • Note that xanthine oxidase can oxidize two different sites on the purine ring system NH O 2 Adenosine C C N Deaminase N N C HN C CH CH HC C HC C O N N N N C Ribose-P Ribose-P N HN C IMP AMP CH HC C N Xanthine Oxidase N H Hypoxanthine O O C N C N HN C HN C C O CH C C C C O N N O N N H H H H GMP Uric Acid Xanthine (2,6,8-trioxypurine) The end product of purine metabolism Uric acid • Uric acid is the excreted end product of purine catabolism in primates, birds, and some other animals. • The rate of uric acid excretion by the normal adult human is about 0.6 g/24 h, arising in part from ingested purines and in part from the turnover of the purine nucleotides of nucleic acids. • The normal concentration of uric acid in the serum of adults is in the range of 3-7 mg/dl. GOUT • The disease gout, is a disease of the joints, usually in males, caused by an elevated concentration of uric acid in the blood and tissues. • The joints become inflamed, painful, and arthritic, owing to the abnormal deposition of crystals of sodium urate. • The kidneys are also affected, because excess uric acid is deposited in the kidney tubules. The uric acid and the gout Hypoxanthine Out of body Xanthine In urine Uric acid ↑ Over 8mg/dl, in the plasma Diabetese nephrosis …… Gout, Urate crystallization in joints, soft tissue, cartilage and kidney Synthesis of Pyrimidine Nucleotides De novo synthesis • shorter pathway than for purines • Pyrimidine ring is made first, then attached to ribose-P (unlike purine biosynthesis) • only 2 precursors (aspartate and glutamine, plus - HCO3 ) contribute to the 6-membered ring • requires 6 steps (instead of 11 for purine) • the product is UMP (uridine monophosphate) Element source of pyrimidine base Step 1: synthesis of carbamoyl phosphate • Carbamoyl phosphate synthetase(CPS) exists in 2 types: • CPS-I, a mitochondrial enzyme, is dedicated to the urea cycle and arginine biosynthesis. • CPS-II, a cytosolic enzyme, used here. It is the committed step in animals. Step 2: synthesis of carbamoyl aspartate ATCase: aspartate transcarbamoylase • Carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step.