BMB 462Exam # 3 Study Guide Lectures: 17 - 26Lecture 17 Recognize the 5 bases and their associated nucleosides, structures, and physical/chemical properties.Nucleotides carry information and energy in the cell. Nucleosides have a pentose sugar and a base attached. If there is also a phosphate on the 5' atom, they're called nucleotides (can have up to 3 phosphates). The base is flat while the sugar is not. The flatness allows the bases tostack in the core of the DNA helix and that makes the DNA very strong. The faces of the base arehydrophobic, making hydrophobic stacking structure. The edges are polar, which is what allows you to make hydrogen bonds and make base pairing to other bases. Depending on what base you have, you can make different hydrogen bonds.Use nucleotide nomenclature.Base Nucleoside Nucleotide Nucleic AcidPurinesAdenine Adenosine DeoxyadenosineAdenylateDeoxyadenylateRNADNAGuanine GuanosineDeoxyguanosineGuanylateDeoxyguanylateRNADNAPyrimidinesCytosine CytidineDeoxycytidineCytidylateDeoxycytidylateRNADNAThymine ThymidineDeoxythymidineThymidylateDeoxythymidylateDNAUracil Uridine Uridylate RNAList nucleotide Properties.Absorption Spectra - All bases absorb light at 260nm; this is due to the conjugated double bond in the bases, which absorb light.pH and tautomerization - pH can change the form of uracil. When pH is low, uracil switches from lactam to lactim formation. The Hydrogen moves from the N to the O on atom 2. This changes the hydrogen bonding (In lactam N is the donor and O is the acceptor, in lactim, N becomes the acceptor).H-bonding and base pairing in DNA - Because information is stored in the hydrogen bonding of DNA it is important to have the right pH b/c changing pH changes H bonding and you will lose the information. AT has 2 H bonds, and GC has 3 so GC is slightly more stable. Otherwise they are almost identical structurally. The H bonding neutralizes thecharge inside the DNA so the inside of the DNA becomes hydrophobic. Then bases stack on each other and create a hydrophobic core that is very stable. The outside of the DNA is hydrophilic.List nucleotide Functions.Building Blocks for DNA and RNA – DNA transmits information from a parent cell to both daughter cells.Information storage and transfer - Information is stored in DNA and that is replicated andtransferred through transcription into RNA. RNA is then translated into proteins.Energy Carriers - Ribo-nucleotides can also be used as energy carriers. Mostly true for ATP and GTP. So they can be building blocks or energy carriers. Phosphoanhydride bondsare very high in energy so the cell to drive a lot of reactions uses them.Coenzyme “handles” - i.e. CoA, NAD and FAD all have nucleotides included in them.Signaling Molecules - i.e. cAMP. It's a signaling molecule inside the cell that acts as a secondary messenger. (Primary signals, i.e. hormones, are usually outside of the cell).Compare and contrast salvage, de novo purine synthesis, and de novo pyrimidine synthesis.Purines Pyrimidines Salvage-Use PRPP – Synthesis starts with PRPP and atoms are added to buildthe base-Atom sources for bases: amino acids, CO2, and THF-The bases are built attached to ribose.-Intermediate = Inosotate-Branched pathway (Inosotate either makes AMP or GMP)-Regulation: feedback inhibition of the first step by IMP intermediate, AMP, GMP.-Use PRPP – Synthesis starts with PRPP and atoms are added to build the base.-Atom sources for bases: amino acids, CO2, and THF.-Orotate is first built and attached toPRPP. Orotate is then attached to ribose and then is converted by phosphoribosyl transferase into 2 pyrimidines.-Intermediate = Orotate-Linear pathway (orotate-UTP-CTP)-Regulation: feedback inhibition of the first step by CTP.-Bases are constructed fromintermediates produced in the degradative pathway of nucleotides.--Useful for cells that can’t do de novo synthesis.-Purines: phosphoribosyl transferases add PRPP to bases.-Pyrimidines: Uridine phosphorylase adds ribose-1-phosphate to free uracil to make UMP.Lecture 18Describe the atom sources and reactions used in de novo nucleotide biosynthesis.Sources of atoms for de novo synthesisa. Purine - i. Nitrogen - 3 nitrogen atoms come from Glutamine and aspartate (amidotransferases), the fourth is from glycine.ii. Carbon – THF in the form of formate (THF is common source of Carbon) and, more rarely, CO2 can be a carbon sourceiii. Glycine donates part of backboneb. Pyrimidine – i. Aspartate donates part of backbone and the rest comes from carbamoyl phosphate (carbamoyl phosphate comes from carbamoyl synthase.)c. PRPP – aka Phosphoribosylpyrophosphate - Comes from pentose phosphate pathway which produces ribose 5-phosphateII. Reactionsa. Phosphoribosyltransferases - Attach a base to sugar, to PRPPExplain the interconversion of nucleotide mono-, di-, and tri-phosphates.The enzyme that converts NMP to NDP - nucleoside monophosphate kinases (family of 4enzymes). The enzymes are specific for the base (so there's one for A, G, C, and U) but not specific for the sugars (can convert ribose and deoxyribose).They use ATP to attach phosphate. i.e. adenylate kinase uses ATP to convert AMP to ADP. In this instance you actually make two ADPs because the ATP is convertedto ADP as well. The ATP then has to be regenerated.Nucleoside Diphosphate Kinase converts all the different dinucleotides to trinucleotides. ATP is again usually the donor and is converted to ADP.Describe reduction of ribonucleotides to deoxyribonucleotides.To make DNA, the cell needs to convert all NDPs to dNDPs. This is done by ribonucleotide reductase, which uses e- from NADPH to reduce the NDPs; the e- are transferred by thioredoxin. Radical e- are used to catalyze the reaction. Ribonucleotide reductase reduces the 2'C by attacking 3'C and making it more reactive.The enzyme has 2 subunits and subunit one has reactive thio groups attached to serine. The hydrogen atoms come from NADPH and therefor need to be regenerated. An active site radical on the R2 subunit is what causes the ribonucleotide radical to form on the 3' carbon. That radical is what allows the chemistry to occur on the 2' C. The radical allows the 2' hydroxyl to attack the thio group and protonate the 2'-hydroxyl. The protonated hydroxyl can then leave as water. When water leaves, it forms a carbocation. The radical stabilizes the carbocation and the other SH group can now react with the 2' and reduce it. Describe dNTP synthesis.Ribonucleotide
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