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Biochemistry Ch 25 – Nucleotide Biosynthesis294513074295438404074295208026074295-143510109855Bases – A, G, T, U, CPurines – A, GPyrimidines – C, U, TNucleoside – base coupled to 5 C ribose or deoxyribose sugar via β-glycosidic linkageRibonucleosides – adenosine, guanosine, uridine, cytidineDeoxyribonucleosides – deoxyadenosine, deoxyguanosine, thymidine, deoxycytidineNucleotide – nucleoside with phosphate attachedRibonucleotides – adenylate, guanylate, uridylate, cytidylateDeoxyribonucleotides – deoxyadenylate, deoxyguanylate, thymidylate, deoxycytidylate Nucleotide rolesPrecursors of RNA & DNAATP – adenine nucleotide used as universal energy currencyGTP – guanine nucleotide, serves as energy source for more select processesUDP-glucose – nucleotide derivative used in biosyntheses such as glycogen synthesisCyclic nucleotides – act as second messengers in signal transduction (e.g., cyclic AMP)General pathway of de novo synthesisPyrimidines – bases are built as individual molecules, then they are linked to activated ribosePurines – bases are assembled while bound to activated riboseDeoxyribonucleotides are made by reduction of ribonucleotidesThymine (a deoxy base) is made by modification of deoxy uracil nucleotide (dUMP)Salvage pathway – a base is reattached to an activated ribose de novo pathway – base itself is synthesized from simpler starting materials (amino acids, CO2), requiring ATP hydrolysis and an activated ribose5-Phosphoribosyl-1-pyrophosphate (PRPP) – a form of ribose activated to accept nucleotide bases- Bases are added at C1, the pyrophosphate at C1 is what makes it active bc it is a good leaving group and can be further hydrolyzed to make rxn irreversible- Phosphates are added at C513709655715Pyrimidine de novo synthesisRing is synthesized first, then it is attached to PRPPStep 1 – Carbamoyl phosphate (remember it from start of urea cycle)Synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process catalyzed by carbamoyl phosphate synthetase (CPS), requiring 2 ATPs:Source of NH3 is primarily glutamine side chain, not free ammoniaCPS binds glutamine at one end and hydrolyzes it to get NH3 which is funneled through the interior of CPS to the two ATPase sites, “substrate channeling”, remaining in interior of enzyme and avoiding contact of reactive species with H2O; carbamoyl-P released at other endStep 2 – OrotateCarbamoyl phosphate reacts with aspartate, catalyzed by aspartate transcarbamoylaseIntermediates are cyclized and oxidized with NAD to yield orotateStep 3 – OrotidylateOrotate couples to PRPP to form orotidylate, a pyrimidine nucleotide (OMP)The enzyme that catalyzes this addition is pyrimidine phosphoribosyltransferaseStep 4 – Uridylate (uracil nucleotide)Orotidylate (OMP) is converted to uridylate (UMP) by decarboxylation UMP is used for RNA synthesis and is precursor of cytidine and thymidineUMP can be converted as needed to UDP & UTPNucleoside monophosphates are converted into diphosphates by specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl-group donor:UMP + ATP UDP + ADP via UMP kinaseNucleoside diphosphates are converted into triphosphates (or the reverse) by a general nucleoside diphosphate kinase:UDP + XTP UTP + XDP (swaps 3rd P from one to another)Conversion of uracil to cytosineOnly occurs after UMP is converted to UTP, as explained above, because the enzyme only works on UTP (takes UTP CTP)Uracil carbonyl group is replaced by amino group (from glutamine), a reaction that requires ATP hydrolysisPyrimidine synthesis overview:(1) Synthesis of carbamoyl phosphate from bicarbonate and Gln-derived ammonia(2) Formation of orotate ring from carbamoyl phosphate & aspartate(3) Coupling to activated ribose PRPP to form orotidylate OMP(4) Decarboxylation of OMP to form uritidylate (UMP)(5) Phosphotransfer to UMP to form UTP(6) Amination of UTP to form CTP* conversion to dNTPs discussed later *114236576200Purine de novo synthesisPurines are synthesized piece by piece while attached to the ribose frameworkStep 1 – Amination of ribosePPi3088640392430Initial committed step: displacement of pyrophosphate from PRPP by glutamine-derived ammonia, resulting in 5-phosphoribosyl-1-amineNH3246316551435Next several stepsThe next six steps are analogous reactions: displacement of phosphates by aminesEach step consists of activation of a carbon-bound oxygen atom by phosphorylation, followed by displacement of the phosphoryl group by ammonia or an amine group as a nucleophile:We just need to know some of the donors in the reactions: Gly, Gln, Asp, formyl-THF, CO2Know that fumarate is released, and both rings are closed, resulting in inosinate (IMP)IMP is a purine used as “mother molecule” to produce AMP and GMP:Inosinate yields adenylate and guanylateAmination of IMP (via Asp-derived amino group) with release of fumarate results in adenylate, requiring GTP as the phosphate donorOxidation of IMP (with NAD+) and amination (via Gln-derived amino group) results in guanylate, requiring ATP as the phosphate donorNote that the synthesis of AMP requires GTP, whereas the synthesis of GMP requires ATP.This reciprocal use of nucleotides by the two pathways creates an important regulatory opportunity:Low GTP means production of adenylate will be reduced, increasing the production of guanylateBut adenylate is the precursor for ATP, so as less adenylate is produced that means less ATP is now available to make GMPLow ATP means production of guanylate will be reduced, increasing the production of adenylateBut guanylate is the precursor for GTP, so as less guanylate is produced that means less GTP is available to make AMPAnd so the regulatory cycle continues…Purine synthesis overview:(1) Amination of PRPP via displacement of its PPi with amine group(2) Multiple phosphorylations via ATP which activate CO groups for displacement with amine group. Sources of atoms include Gly, Gln, Asp, THF-formyls, CO2(3) Resulting IMP is aminated to yield AMP and GMP(4) As with the pyrimidines, the AMP and GMP are easily converted to ATP and GTP via phosphotransfer by specific and general kinases580390-81280Synthesis of deoxyribonucleotidesSo far we have produced purines and pyrimidines in the form of ribonucleotides (UTP, CTP, ATP, GTP)The precursors of DNA, deoxyribonucleotides, are formed by the reduction of ribonucleotides, specifically, the reduction of the 2’-hydroxyl group on the ribose to a hydrogenThe reductant used is NADPH, the enzyme that catalyzes the reduction is ribonucleotide reductase – the same enzyme will bind and reduce all four ribonucleotides (binds them as diphosphates, NDPs)The initial products of this reaction are therefore dNDPs (dUDP, dCDP, dADP, dGDP)Thus, further processing is required to convert the uridylate to thymidylateDNA contains thymine, the methylated analog of uracilThis methylation is catalyzed by thmyidylate synthase which requires deoxyuridylate (dUMP) as its substrate (so the initial product, dUDP, will transfer its P to form dUMP)Methylation of dUMP (donor is methylene-THF) results in thymidylate (TMP) & dihydrofolateDihydrofolate can be reduced with NADPH via dihydrofolate reductase to regenerate the THFThymidylate synthesis blocked by anticancer drugsRapidly dividing cells require an abundant supply of thymidylate for synthesis of DNAInhibition of TMP synthesis machinery is thus a target for cancer drug therapy, because most of your cells won’t be affected by decreased TMP production, but rapidly dividing cancer cells willFlurouracilanticancer drug that blocks TMP synthesis via inhibition of thymidylate synthaseForms fluorodeoxyuridylate (F-dUMP) which irreversibly binds thymidylate synthase and is not released from the enzymeThis is an example of “suicide inhibition” in which the enzyme is trapped in a form that cannot proceed down the reaction pathwayDihydrofolate analogsSynthesis of TMP can also be blocked by inhibiting the regeneration of THFAnalogs of dihydrofolate (aminopterin & methotrexate) are competitive inhibitors of dihydrofolate reductase (the enzyme that is used to regenerate THF from dihydrofolate)Inhibition of dihydrofolate reductase thus inhibits the regeneration of THF, which inhibits the production of TMP (because TMP synthesis requires methylation of dUMP by means of methylene-THF)Regulation of nucleotide biosynthesisNucleotide biosynthesis is regulated by feedback inhibition in a manner similar to regulation of aa biosynthesis: the regulatory pathways ensure that the various nucleotides are produced in the required quantities and ratiosPyrimidine regulationThe step in pyrimidine synthesis where aspartate and carbamoyl phosphate form the orotate ring is catalyzed by aspartate transcarbamoylaseAspartate transcarbamoylase is inhibited by CTP, the final product of pyrimidine synthesisAspartate transcarbamoylase is stimulated by ATPPurine regulationCommitted step in purine nucleotide synthesis is the amination of PRPP, which is feedback-inhibited by IMP, AMP, and GMPThe two paths of inosinate, to AMP or GMP, are also feedback inhibited, so GMP inhibits the conversion of inosinate to GMP; AMP inhibits the conversion of inosinate to AMPAlso remember the inosinate pathways are regulated by GTP and ATP (already discussed – because GTP is required for AMP synthesis, and ATP is required for GMP synthesis)Regulation of deoxyribonucleotidesThe synthesis of dNTPs is controlled by the regulation of ribonucleotide reductase (the enzyme that catalyzes the reduction of ribonucleotides to yield deoxyribonucleotides)Each subunit in the ribonucleotide reductase enzyme has two allosteric binding sites, one of which regulates overall activity, the other regulates substrate specificityOverall catalytic activity is diminished by the binding of dATP, which is a signal of overabundance of all the deoxyribonucleotides. The binding of ATP reverses this feedback inhibition. Specificity for nucleotides is determined by each dNTP binding to the specificity allosteric site on the enzyme, each stimulating the binding of the other (non-self) nucleotides. This complex pattern of regulation supplies the appropriate balance of the four deoxyribonucleotides needed for the synthesis of DNA.Nucleotide metabolic problemsTurnover of nucleotidesDephosphorylation of nucleotides by nucleotidasesCleavage to bases and ribose-1-P by nucleoside phosphorylasesRibose-1-P is isomerized by phosphoribomutase to yield ribose-5-P, a substrate in the synthesis of PRPPSome of the bases are reused to form nucleotides via salvage pathways, others are degraded to products that are excretedDeficiency in enzymes can disrupt these breakdown pathways, leading to various pathological conditionsFailure of deamination of adenosine is associated with severe combined immunodeficiency (SCID- the bubble boy disease) – an ineffective immune system due to loss of T-cell activityBreakdown of purines results in uric acid which is converted to urate to be excreted in urine. Higher than usual purine catabolism results in elevated levels of urate in the bloodstream, which crystallize and resulting the joint disease goutDisruption of the salvage pathway via genetic mutation of guanine phosphoribosyltransferase (the enzyme that couples guanine to PRPP) results Lesch-Nyhan syndrome (compulsive self-destructive behavior) and mental deficiencies. The elevated levels of PRPP lead to an increase in the rate of purine biosynthesis by the de novo pathway, and accordingly, an overproduction of urate.Folic acid deficiency can lead to neural tube defects (spina bifida). More folate derivates may be needed for the synthesis of DNA precursors when cell division is frequent and substantial amounts of DNA must be synthesized (growth & development during pregnancy) . ................
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