BMB 462 Lecture 18 Outline of Last Lecture I. Nucleotide structure and nomenclaturea. i.e. dAMPII. Nucleotide propertiesIII. Nucleotide FunctionIV. Nucleotide Metabolisma. Regulation of metabolismOutline of Current Lecture I. Sources of atoms for de novo synthesisII. Function and Defects of PhosphoribosyltransferasesIII. Defects in Nucleotide BreakdownIV. Nucleoside mono-, di-, and tri- phosphate conversionV. Ribonucleotide ReductaseVI. dNTP SynthesisVII. Thymidylate SynthesisVIII. Inhibition of dNTP SynthesisCurrent LectureConcepts to remembers from previous courses/lectures:-I. 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 phosphate1. 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 Phosphoribosylpyrophosphatei. A central molecule in synthesisii. Comes from pentose phosphate pathway which produces ribose 5-phosphateiii. Then ATP donates the 2 phosphates, which are attached to C1' and that makes it very reactive so other atoms can be attached to itII. Function and Defects of Phosphoribosyltransferasesa. Functioni. Attach a base to sugar, to PRPPii. The enzyme that helps with this is orotate phosphoribosyl transferaseiii. There are also phosphoribosyl transferases that work in salvage and transfer other bases to PRPPb. Defects – Lesch-Nyhan Syndromei. 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 knownii. Children with Lesch-Nyhan syndrome are also known to self mutilateiii. The disease is related to central nervous system.III. Defects in Nucleotide Breakdowna. 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-SCIDSi. Mutation in adenosine deaminase leads to ADA-SCIDS, an immuno-deficient diseaseii. AMP accumulates, so dATP also accumulates and that leads to a down regulation of the other dNTPsiii. The reduction in dNTPs means the body doesn't have enough nucleotidesto 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 dNTPsc. Gouti. 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 becomesvery painful.IV. Nucleoside mono-, di-, and tri- phosphate conversiona. Nucleoside Monophosphate Kinasesi. Need to make NDPs and NTPs from NMPs1. 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 phosphatec. 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 wellii. ATP is used in cell by all kinases to donate phosphate and needs to be regeneratedb. Nucleoside Diphosphate Kinasei. 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 ADPV. Ribonucleotide Reductasea. Now that we've made all the NTPs, the molecules can be used to make RNA, or for signaling, energy storage, or cofactorsb. Electrons from NADPH reduce NDPsi. 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 thioredoxini. The enzyme (a reductase) uses radical chemistry – it uses a radical e- to catalyze the reactionb. Ribonucleotide reductase reduces the 2'C by attacking 3'C and making it more reactivec. 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' C3. The radical allows the 2' hydroxyl to attack the thio group and protonate the 2'-hydroxyl. The protonated hydroxyl can then leaveas 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 NADPH4. The new dNDP now leaves active site and a new NDP can bindVI. dNTP Synthesisa. 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 whichone 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 activityd. Regulation of Overall Activity i. The primary regulatory site regulates overall enzyme activity. It does that by binding ATP or dATP1. 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 Specificityi. Substrate specificity regulatory site regulates substrate specificity; determines which substrate is
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