Nucleic Acid Metabolism Lecture PDF

Dr. Ardina Grüber Nanyang Technological University School of Biological Sciences Division of Structural Biology and Biochemistry Singapore 637551 [email protected]  How does the seed know what kind of plant...

Dr. Ardina Grüber Nanyang Technological University School of Biological Sciences Division of Structural Biology and Biochemistry Singapore 637551 [email protected]  How does the seed know what kind of plant to become? How does a fertilized egg know how to grow into a human being? And as the fertilized egg grows into a developing embryo, how does any given cell know to become part of a finger, a liver, or a heart? The answer to these and a multitude of other fundamental questions about all living organisms reside in the biological molecules known as nucleic acids. Mathews, van Holde, Ahern: Biochemistry 3rd edition Nucleic acids are the chemical carriers of an organism’s genetic information. There are two classes of nucleic acids: Deoxyribonucleic acid, DNA (the storage and transmission of biological information). Ribonucleic acid, RNA (broader range of functions, and several classes are found in cells). Ribosomal RNAs (rRNAs) - components of ribosomes, the complexes that carry out the synthesis of proteins. Messenger RNAs (mRNAs) - intermediaries, carrying genetic information from one or a few genes to a ribosome. Transfer RNAs (tRNAs) - adapter molecules that translate the information in mRNA into a specific sequence of amino acids. Nucleic acids are polynucleotides, and each nucleotide has three parts: - a five-membered cyclic monosaccharide - a phosphate group (-OPO32-) - and a nitrogen-containing cyclic compound known as a nitrogenous base  Pyrimidine Purine Garrett, Grisham: Biochemistry 4th edition  Thymidine Nucleosides are named by adding the ending -idine to the root name of a pyrimidine or -osine to the root name of a purine. The common nucleosides are thus cytidine, uridine, thymidine, adenosine, and guanosine. Garrett, Grisham: Biochemistry 4th edition  In mammals, adenosine functions as an autacoid, or “local hormone.” Caffeine is an alkaloid. It promotes wakefulness by blocking the interaction of extracellular adenosine with its neuronal receptors. '“Autacoids are a locally produced modulating factors, influencing locally the function of cells and/or tissues, which are produced on demand and which subsequently are metabolized in the same cells and/or tissues".(Keppel Hesselink, JM., Auton Autacoid Pharmacol. 35: 51–8, 2015) Garrett, Grisham: Biochemistry 4th edition – Precursors of DNA and RNA: purines and pyrimidines – Carriers of chemical energy: ATP, GTP – Cofactors: NAD, FAD, CoA, S-adenosyl methionine, – Activated intermediates: UDP-glucose – Second messengers: cAMP, cGMP  The successive nucleotides of both DNA and RNA are covalently linked through phosphate- group “bridges,” in which the 5-phosphate group of one nucleotide unit is joined to the 3- hydroxyl group of the next nucleotide, creating a phosphodiester linkage. All the phosphodiester linkages have the same orientation along the chain, giving each linear nucleic acid strand a specific polarity and distinct 5 and 3 ends. Lehninger: Biochemistry 4th edition  Other Functions of Nucleotides Nucleotides carry chemical energy in cell ATP/ADP/AMP – energy metabolism, pumping materials across the membrane, muscle contraction, etc. UTP – sugar interconversion and synthesis of oligosaccharides: UTP in animals, ATP in bacteria and plants-synthesis of storage polysaccharides (glycogen and starch) GTP – forming GDP-activated sugars-synthesis of complex carbohydrates – forming peptide chain - synthesis of proteins – interacting with G proteins – transmission of information across cell membranes Lehninger: Biochemistry, 4th edition Other Functions of Nucleotides Adenine nucleotides are components of many enzyme cofactors Adenosine part does not participate directly in the primary function Removal of adenosine part from cofactor - drastic reduction of cofactor activities Lehninger: Biochemistry, 4th edition  Other Functions of Nucleotides Some nucleotides are regulatory molecules cyclic AMP – metabolic control, especially modulation of protein phosphorylation, which modifies enzymes properties – often acts as a “second messenger” in tandem with G proteins/GTP system cyclic GMP – vision and metabolic controls (p)ppGpp – an alarmone produced in bacteria in response to a slowdown in protein synthesis during amino acid starvation. - inhibits synthesis of the rRNA and tRNA Lehninger: Biochemistry, 4th edition Two types of biosynthetic pathways: – Salvage pathways: recycle of free bases and nucleotides released from nucleic acid breakdown – De novo pathways: using metabolic precursors: amino acids, ribose 5-phosphate, CO2, and NH3 Important precursors: – PRPP (phosphorybosyl pyrophosphate) – Carbamoyl phosphate – Amino acids: glycine, aspartate, and glutamine Important distinctive features: – Purine ring is built atom by atom on the ribose base – Ribose base is attached after the pyrimidine ring is formed  1948 John Buchenan and Robert Greenberg exploited the fact that birds excrete excess of nitrogen principally in the form of uric acid. Tracing the metabolic source of the various atoms in this end product, they showed that the nine atoms of the purine ring system are contributed by different molecules. Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme  Most de novo synthesis occurs in the liver and the nitrogenous bases and nucleosides are then transported to other tissues by red blood cells. Purines are synthesized as ribonucleotides, with the initial purine synthesized being inosine monophosphate (IMP). Adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are each derived from IMP in two-step reaction pathways. Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  5-Phospho-α-D-ribosyl -1- pyrophosphate (PRPP) is a key intermediate in de novo synthesis of both purine and pyrimidine nucleotides. Moran, Horton, Scrimgeour, Perry: Principles of Biochemistry 5th edition  The first step in purine biosynthesis is committed step of the pathway. The purine base is built on the ribose moiety. 5-Phosphorybosylamine is highly unstable. The availability of the substrate PRPP is a major determinant of the rate of this reaction. Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  The second step is the addition of three atoms from glycine. The ATP is required for the condensation of the glycine carboxylic acid group with the 1′- amino group of phosphoribosyl 1-amine. Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  3. 4. 5. 6. 7. Adenine nucleotides Guanine nucleotides Marks Basic Medical Biochemistry: A clinical Approach, 4th edition Lehninger: Biochemistry, 4th edition AMP and GMP are converted by successive phosphorylation reactions into their metabolically prominent triphosphate forms, ATP and GTP. The first phosphorlyation, to give the nucleoside diphosphate forms, is carried out by two base-specific, ATP- dependent kinases, adenylate kinase and guanylate kinase. Adenylate kinase: AMP + ATP → 2 ADP Guanylate kinase: GMP + ATP → GDP + ADP These nucleoside monophosphate kinases also act on deoxynucleotide monophosphates to give dADP or dGDP. Oxidative phosphorylation is primarily responsible for the conversion of ADP into ATP. ATP then serves as the phosphoryl donor for synthesis of the other nucleoside triphosphates from their corresponding NDP’s in a reaction catalyzed by nucleoside diphosphate kinase, a nonspecific enzyme. For example, GDP + ATP ↔ GTP + ADP  Reutilization of purine and pyrimidine bases. Relationships between nucleic acid catabolism (blue) and resynthesis of nucleotides by salvage pathways (red). One of the primary salvage pathways consists of a single reaction catalyzed by adenosine phosphoribosyltransferase, in which free adenine reacts with PRPP to yield the corresponding adenine nucleotide: Free guanine and hypoxanthine (the deamination product of adenine are salvaged in the same way by hypoxanthine-guanine phosphoribosyltransferase. Mathews, van Holde, Appling, Anthony-Cahill: BIOCHEMISTRY 4rd edition  Regulation of purine synthesis occurs at several sites. Four key enzymes are regulated: 1. PRPP synthetase, 2. amidophosphoribosyl transferase, 3. adenylosuccinate synthetase, and 4. IMP dehydrogenase. The first two enzymes regulate IMP synthesis; the last two regulate the ---------------------- production of AMP and GMP, respectively. ---------------- GDP ADP GTP ------- ATP ---- --------------- Garrett, Grisham: Biochemistry 4th edition  The general strategy is conversion of the nucleotides to nucleosides catalyzed by intracellular nucleotidases. Nucleosides are then degraded by the enzyme purine nucleoside phosphorylase (PNP) to release the purine base and ribose-l- phosphate. The PNP products are merged into xanthine by guanine deaminase and xanthine oxidase, and xanthine is then oxidized to uric acid by this latter enzyme. Xanthine oxidase is present in large amounts in liver, intestinal mucosa and milk. Its oxidized hypoxanthine to xanthine and xanthine to uric acid. Garrett, Grisham: Biochemistry 4th edition  Most of the mammals posses active ureate oxidase to convert uric acid to allantoin. Chronic elevation of blood uric acid levels well beyond the already high levels is called hyperuricemia. Although the biochemical reasons for hyperuricemia vary, the conditions goes by the single clinical name gout. Mathews, van Holde, Appling, Anthony-Cahill: BIOCHEMISTRY 4rd edition Gout is the medical condition characterized by excessive amounts of uric acid in the body. PRPP synthetase and PRPP amidotransferase Defects in these enzymes may render it insensitive to feedback inhibition by purine nucleotides. Thus, purine nucleotides are overproduced, leading to excessive uric acid synthesis and gout. Hypoxanthine-Guanine phosphoribosyltransferase (HGPRT) is a salvage pathway enzyme for purine metabolism. Some gouts are successfully treated by allopurinol, a substrate analog inhibitor of xanthine oxidase.  Lesch-Nyhan Syndrome - completely absence or severe deficiency of the HGPRT enzyme activity leads to severe gouty arthritis. The structural gene for HGPRT is located on the X-chromosome, and this disease is a congenital, recessive, sex-linked trait manifested only in males. Although, HGPRT may play minor role in purine metabolism, its absence has profound consequences: De novo purine biosynthesis is dramatically increased, and uric acid level in the blood are elevated. http://dx.doi.org/10.1053/j.ajkd.2008.09.011 HPRT - Hypoxanthine-Guanine phosphoribosyltransferase APRT - Adenine phosphoribosyltransferase  In contrast to purine, pyrimidines are not synthesized as nucleotide derivatives. The pyrimidine ring system is constructed before a ribose-5-P moiety is attached. Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme The pyrimidine synthesis begins with formation of carbamoyl phosphate, a reaction catalyzed by carbamoyl phosphate synthetase II (CPS II). Carbamoyl phosphate is formed from ATP, bicarbonate, and the amide nitrogen from glutamine. Marks Basic Medical Biochemistry: A clinical Approach, 4th edition  De novo biosynthesis of pyrimidine ribonucleotides 1. Carbamoyl phosphate synthetase II 2. Aspartate transcarbamoylase 3. Dihydroorotase 4. Dihydroorotate dihydrogenase 5. Orotate phosphoribosyl-transferase 6. Orotidylate decarboxylate 7. ATP-dependent nucleoside monophosphate kinase 8. Nucleoside diphosphate kinase 9. CTP synthetate © 2016 Pearson Education, Ltd.  ATCase Aspartate transcarbamoylase (ATCase) CPS II Carbamoyl phosphate synthetase II Garrett, Grisham: Biochemistry 4th edition  Pyrimidine catabolism results in degradation of the pyrimidine ring to products reminiscent of the original substrates, aspartate, CO2, and ammonia. Catabolism of cytosine and uracil yields -alanine, ammonium ion, and CO2. Catabolism of thymine yields -aminoisobutyric acid, ammonium ion, and CO2. Garrett, Grisham: Biochemistry 4th edition  Ribonucleotides are precursors of deoxy- ribonucleotides. The pathway involves reduction of ribose to deoxyribose and conversion of uracil to thymine. Ribose to deoxyribose: replace C-2 hydroxyl ion by a hydride ion. The enzyme, ribonucleotide reductase, reduces all four common ribonucleotide substrates. As a result, the enzyme is oxidized. Garrett, Grisham: Biochemistry 4th edition  Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides All four ribonucleotides are reduced by RNR RNR uses a free radical mechanism, though three classes are found in nature: – Class I, widespread, uses a catalytic Tyr radical – Class II uses B12 coenzyme to generate the radical – Class III, which functions only under anaerobic conditions, uses S- adenosylmethionine to generate a glycine radical RNR is an α2β2 heterodimer in E. coli, yeast, and humans The Tyr radical is in the small β- subunit, some 35 Å from the active site in the large α-subunit  Dimeric thioredoxin reductase with bound ligands: FAD and NADP. Each monomer is associated with a thioredoxin molecule (yellow). Garrett, Grisham: Biochemistry 4th edition  NADPH is the ultimate source of electrons for RNR These electrons are shuttled through either thioredoxin (red arrows) or glutaredoxin (blue arrows).  RNR activity and specificity are strictly regulated RNR has two classes of control sites, activity sites and specificity sites, to ensure optimally balanced pools of DNA precursors are maintained. © 2016 Pearson Education, Ltd.  Overview of deoxyribonucleoside triphosphate synthesis ▪ Most cells contain 5 to 10 times more RNA than DNA ▪ Ribonucleotides serve multiple metabolic roles, while deoxyribonucleotides only make up DNA ▪ Two specific processes make up dNTP biosynthesis: ▪ Origin of deoxyribose ▪ Origin of the thymine methyl group © 2016 Pearson Education, Ltd.