BMB 462 Lecture 18 Outline of Last Lecture I Nucleotide structure and nomenclature a i e dAMP II Nucleotide properties III Nucleotide Function IV Nucleotide Metabolism a Regulation of metabolism Outline of Current Lecture I Sources of atoms for de novo synthesis II Function and Defects of Phosphoribosyltransferases III Defects in Nucleotide Breakdown IV Nucleoside mono di and tri phosphate conversion V Ribonucleotide Reductase VI dNTP Synthesis VII Thymidylate Synthesis VIII Inhibition of dNTP Synthesis Current Lecture Concepts to remembers from previous courses lectures I Sources of atoms for de novo synthesis a 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 source iii Glycine donates part of backbone b Pyrimidine i Aspartate donates part of backbone and the rest comes from carbamoyl phosphate 1 carbamoyl phosphate from carbamoyl synthase These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute c PRPP aka Phosphoribosylpyrophosphate i A central molecule in synthesis ii Comes from pentose phosphate pathway which produces ribose 5phosphate iii Then ATP donates the 2 phosphates which are attached to C1 and that makes it very reactive so other atoms can be attached to it II Function and Defects of Phosphoribosyltransferases a Function i Attach a base to sugar to PRPP ii The enzyme that helps with this is orotate phosphoribosyl transferase iii There are also phosphoribosyl transferases that work in salvage and transfer other bases to PRPP b Defects Lesch Nyhan Syndrome i If there is a mutation in Hypoxanthine guanine phosphorybosyl transferase HGPRT then you develop Lesch Nyhan which is associated with mental retardation The mechanism is not well known ii Children with Lesch Nyhan syndrome are also known to self mutilate iii The disease is related to central nervous system III Defects in Nucleotide Breakdown a The bases are either converted via salvage to be reused or excreted as uric waste Defects in this result in b Adenosine Deaminase and ADA SCIDS i Mutation in adenosine deaminase leads to ADA SCIDS an immunodeficient disease ii AMP accumulates so dATP also accumulates and that leads to a down regulation of the other dNTPs iii The reduction in dNTPs means the body doesn t have enough nucleotides to make DNA and without that ability the cells can t divide and replicate iv This makes it difficult for the immune system because for it to work lymphocytes have to rapidly proliferate but they cannot without dNTPs c Gout i Failure to excrete uric acid and the resulting uric acid build up leads to gout Though the mechanism is not entirely known ii Uric acid is not excreted and begins crystallizing in the joints and becomes very painful IV Nucleoside mono di and tri phosphate conversion a Nucleoside Monophosphate Kinases i Need to make NDPs and NTPs from NMPs 1 The enzyme that converts NMP to NDP nucleoside monophosphate kinases family of 4 enzymes a 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 b They use ATP to attach phosphate c i e adenylate kinase uses ATP to convert AMP to ADP In this instance you actually make two ADPs because the ATP is converted to ADP as well ii ATP is used in cell by all kinases to donate phosphate and needs to be regenerated b Nucleoside Diphosphate Kinase i Have NDP and need to convert it to NTP Nucleoside Diphosphate Kinase converts all the different dinucleotides ii ATP is again usually the donor and is converted to ADP V Ribonucleotide Reductase a Now that we ve made all the NTPs the molecules can be used to make RNA or for signaling energy storage or cofactors b Electrons from NADPH reduce NDPs i They are transferred from NADPH to Ribonucleotide Reductase by Thioredoxin or Glutaredoxin 1 To make DNA the cell needs to convert all NDPs to dNDPs This is done by ribonucleotide reductase a Uses e from NADPH to reduce the NDPs the e are transferred by thioredoxin i The enzyme a reductase uses radical chemistry it uses a radical e to catalyze the reaction b Ribonucleotide reductase reduces the 2 C by attacking 3 C and making it more reactive c 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 2 Active site radical on the R2 subunit The Radical probably originally goes from a Tyrosine to a Cysteine And this 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 3 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 a dNDP is formed In the process the thiol groups have been oxidized and formed a disulfide bond These need to be regenerated Thioredoxin does this using H from NADPH 4 The new dNDP now leaves active site and a new NDP can bind VI dNTP Synthesis a Ribonucleotide Reductase is a dimer so it contains 2 catalytic subunits and 2 active sites Chemistry can happen in both sites b The two regulatory sites determine what is converted substrates are ADP UDP GDP CDP and all can bind to the active site The regulatory sites determine which one binds though i The regulatory sites bind allosteric effectors ATP dATP dGTP dTTP c If there s a lot of dATP that means energy is low and there is little activity d Regulation of Overall Activity i The primary regulatory site regulates overall enzyme activity It does that by binding ATP or dATP 1 When ATP is bound the enzyme is active and converts the substrates Binding ATP measures that the cell has a lot of energy and so can make DNA and divide 2 If there s a lot of dATP and that s what binds it means that energy is low so the enzyme is inactive The enzyme essentially measures energy level in cell e Regulation of Substrate Specificity i Substrate specificity regulatory site regulates substrate specificity determines which substrate is to be converted Determined by binding either ATP dATP dGTP dTTP ii Essentially it measures which nucleotides are already present in the cell 1
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