Nucleotide Structure and Synthesis Lecture (PDF)

Nucleotide Structure and Synthesis Wednesday October 30, 2024  Goals Recognize nitrogenous bases (purines and pyrimidines), nucleosides and nucleotides Describe the precursor molecules required to form purines and pyrimidines by the de novo pathway Pu...

Nucleotide Structure and Synthesis Wednesday October 30, 2024  Goals Recognize nitrogenous bases (purines and pyrimidines), nucleosides and nucleotides Describe the precursor molecules required to form purines and pyrimidines by the de novo pathway Purine ring is built from scratch at the C1 position of PRPP – (don’t need to know order of assembly) – first make IMP Pyrimidne ring is built from scratch, then attached to PRPP – first make OMP Describe how other nucleotides are made from IMP and OMP Describe mechanisms regulating nucleotide synthesis Describe how nucleotide monophosphates are converted to nucleotide di- and tri phosphates Describe the synthesis of nucleotides by the salvage pathway Nitrogenous bases Recall purines and pyrimidines from lecture 1 thway ide i.e. small biomolecules e nucleot alvage pa At the time we considered 2 purines - adenine t in purin - guanine ion and s and 3 pyrimidines - thymine Importan degradat - cytosine - uracil Introduce here two additional purines - xanthine - hypoxanthine FIGURE 23 The Most Common Naturally Occurring Purines (a) and Pyrimidines (b)  Nucleosides A nucleoside is a biomolecule containing a nitrogenous base (in this case an adenine) covalently attached to the C-1 position of a sugar (in this case ribose) via a β-glycosidic linkage Note the numbering of the sugar, e.g. 2’ position, and the numbering of the base, e.g. 2 position. FIGURE 25 Nucleoside Structure  Nucleotides Recall from lecture 1 that nucleotides contain a nitrogenous base, a sugar and 1, 2 or 3 phosphate groups synt mediate These are nucleotide hesis monophosphates, i.e. 1 phosphate er in pu rtant int group Note the phosphate is attached rine to the 5’ position of the sugar o Imp Note the nomenclature adenosine = nucleoside FIGURE 26 Common Ribonucleotides adenosine-5’-monophosphate = nucleotide deoxyadenosine-5’-monophosphate = nucleotide with deoxyribose sugar Nucleotide synthesis Purine and pyrimidine nucleotides can be synthesized by two pathways - the de novo pathway - built from amino acids, ribose 5-phosphate and CO2 - the salvage pathway - built from free bases and nucleosides generated by nucleic acid breakdown De novo nucleotide synthesis FIGURE 27 PRPP Synthesis Recall that pentose phosphate pathway generates ribose 5-phosphate In both de novo purine and pyrimidine nucleotide synthesis ribose 5-phosphate is the precursor Ribose 5-phosphate is converted to PRPP De novo nucleotide synthesis De novo purine synthesis: 1. Start with PRPP 2. Build purine base on PRPP (at arrow) 3. Product is IMP De novo pyrimidine synthesis: 1. Build base first (orotate) 2. Covalently attach orotate to PRPP (at arrow) 3. Product is OMP  Building a purine base glycine CO2 aspartate N10-formyl-THF N10-formyl-THF formate glutamine formate glutamine The biosynthetic pathway of the first nucleotide synthesized in the de novo pathway. * Don’t worry about the order of addition or exactly which atoms come from which precursor – but know all the components you need to build * This enzyme, glutamine-PRPP amidotransferase, is an important point of regulation Inosine 5’-monophosphate (IMP) is the product of this pathway The nitrogenase base in IMP is hypoxanthine The other purine nucleotides, AMP and GMP are produced by modification of the nitrogenous base of IMP De novo purine nucleotide synthesis M P End product of this series of I reactions is IMP IMP is the precursor for synthesis of AMP and GMP These reactions result in modification of the base of IMP to make AMP and GMP P A MP G M Figure 14.28 Biosynthesis of AMP and GMP from IMP In the first step of AMP synthesis, the amino group of aspartate replaces the C-6 keto oxygen of the hypoxanthine base moiety of IMP. In the second step, the product of the first reaction, adenylosuccinate, is hydrolyzed to form AMP and fumarate. GMP synthesis begins with the oxidation of IMP to form XMP. GMP is produced as the amide nitrogen of glutamine replaces the C-2 keto oxygen of XMP. Note that AMP formation requires GTP and that GMP formation requires ATP. TRUDY MCKEE, JAMES R. MCKEE, Biochemistry, The Molecular Basis of Life 13 © 2020 Oxford University Press Synthesis of AMP from IMP IMP differs from AMP by 1 function group Need nitrogen to convert IMP to AMP Source is aspartate GTP is required to make AMP from IMP!  Synthesis of GMP from IMP IMP differs from GMP by 1 function group Takes 2 reactions First reaction oxidizes IMP to xanthosine monophosphate (XMP) Need nitrogen to convert XMP to GMP Source is glutamine ATP is required to make GMP from IMP Regulation of nucleotide synthesis Why – high cost of energy - ~7 ATP equivalents to make IMP, 1 GTP to make AMP from IMP, 2 ATP to convert AMP to ATP = consumption of 10 ATP equivalents to make 1 ATP molecule de novo Recall important principles of regulation End product feeds back to regulate Often allosteric Regulation occurs at the top of the pathway and at branchpoints Allosteric negative feedback of purine X synthesis Enzyme is allosterically regulated by very high concentrations of IMP, AMP and GMP - not physiological Major site of regulation is allosteric negative feedback of the activity of glutamine-PRPP amidotransferase by IMP, AMP and GMP Adenylosuccinate synthetase is allosterically regulated by negative feedback by AMP IMP dehydrogenase is allosterically regulated by negative feedback by GMP & XMP Balance of synthesis Recall GTP required to make AMP and ATP required to make GMP If GMP is low, GTP will be low, therefore synthesis of AMP is reduced while synthesis of GMP proceeds If AMP is low, ATP will be low, therefore synthesis of GMP is reduced while synthesis of AMP proceeds De novo synthesis of pyrimidines PRPP is a precursor pyrimidine base is synthesized prior to attachment to the ribose sugar Need two nitrogens – one from glutamine and one from aspartate Amine from glutamine is incorporated into carbamoyl phosphate Carbamoyl phosphate is covalently linked to aspartate and the molecule cyclized to produce a pyrimidine, orotate Orotate reacts with PRPP to generate a pyrimidine nucleotide (OMP) FIGURE 31 Pyrimidine Nucleotide Synthesis  Pyrimidine synthesis recap Aspartate Carbamoyl phosphate synthetase II generates carbamoyl phosphate from ATP, HCO3-, and glutamine Carbamoyl phosphate is Carbamoyl covalently linked to aspartate phosphate by aspartate transcarbamoylase FIGURE 32 Origin of Pyrimidine Ring Atoms All three of these enzyme activities are encoded by the same polypeptide – mechanism to efficiently Ring closure is catalyzed by dihydroorotase synthesize the product OMP is converted to UMP by decarboxylation FIGURE 31 iii Synthesis of CTP CTP is generated from UTP by accepting an amide nitrogen from glutamine Pyrimidine synthesis is regulated by negative UMP + ATP UDP + ADP feedback – CTP allosterically inhibits aspartate UDP + ATP UTP + ADP transcarbamoylase, the enzyme linking aspartate and carbamoyl phosphate O O O O O O O- P O P O P O- P O P O P O- O- O- O- O- O- Synthesis of NDP and NTP from NMP AMP + ATP 2 ADP This reaction requires ATP NMP + ATP NDP + ADP This reaction requires ATP NDP + ATP NTP + ADP This reaction requires a phosphate donor – most often ATP but could be GTP, CTP or UTP NMP = AMP, GMP, UMP or CMP, etc Nucleotide synthesis by the salvage pathway FIGURE 27 PRPP Synthesis Since de novo synthesis is energetically expensive there are mechanisms to recycle nitrogenous bases – salvage pathway Bases released from degraded nucleic acids are reused to generate more nucleotides PRPP is a precursor for the salvage pathway Nitrogenous bases are covalently linked to PRPP releasing PPi (irreversible) Salvage pathways are important hypoxanthine-guanine phosphoribosyltransferase (HGPRT) converts hypoxanthine and PRPP to IMP also converts guanine and PRPP to GMP Lesch-Nyhan syndrome – X-linked disease Neurological symptoms – self mutilation, involuntary movements and mental retardation Cause is excess production of uric acid (degradation product of purines) Genetic basis is mutation in HGPRT