1、Cellular Roles of NucleotidesCellular Roles of Nucleotides Energy metabolism(ATP)*Monomeric units of nucleic acids*Regulation of physiological processes Adenosine controls coronary blood flow cAMP and cGMP serve as signaling molecules Precursor function-GTP to tetrahydrobiopternin Coenzyme component
2、s-5-AMP in FAD/NAD+Activated intermediates-UDP Glucose Allosteric effectors-regulate themselves and othersHow I hope to make this at least bearable if not How I hope to make this at least bearable if not mildly interestingmildly interesting Purines and Pyrimidines Synthesis(de novo and salvage pathw
3、ays)Degradation Relevant disease states Relevant clinical applicationsYou are not responsible for any structuresPurines and PyrimidinesPurines and PyrimidinesAdenineGuanineThymine/UracilCytosineTwoPurinesTwoPyrimidinesHNCHNCCNCNCNH2HNCCCHNCOCH3HOHNCCCNCHOHHNH2HNCHNCCNCNCOH2NHSynthesis PathwaysSynthe
4、sis PathwaysFor both purines and pyrimidines there are two means of synthesis(often regulate one another)de novo(from bits and parts)salvage(recycle from pre-existing nucleotides)Salvage Pathwayde novo PathwayMany Steps Require an Activated Ribose Sugar Many Steps Require an Activated Ribose Sugar(P
5、RPP)(PRPP)5de novo Synthesisde novo Synthesis Committed step:This is the point of no return Occurs early in the biosynthetic pathway Often regulated by final product(feedback inhibition)XPurine Biosynthesis(de novo)Purine Biosynthesis(de novo)Atoms derived from:Aspartic acidGlycineGlutamineCO2Tetrah
6、ydrofolateAlso requires4 ATPsPurines are synthesized on the Ribose ringCommitted Step Inhibited byAMP,GMP,IMPXPurine Biosynthesis(de novo)Purine Biosynthesis(de novo)ATPGTP(A bunch of steps you dont need to know)(Inosine Monophosphate)NCHNCCNCNCOHNCHNCCNCNCOH2NHNCHNCCNCNCNH2HFeedbackInhibitionSalvag
7、e Pathway for PurinesSalvage Pathway for PurinesHypoxanthineorGuanine+PRPP=IMP or GMP+PPi Hypoxanthineguanosylphosphoribosyl transferase(HGPRTase)Adenine+PRPP=AMP+PPi Adeninephosphoribosyl transferase(APRTase)Lesch-Nyhan SyndromeLesch-Nyhan SyndromeAbsence of HGPRTaseX-linked(Gene on X)Occurs primar
8、ily in malesCharacterized by:Increased uric acidSpasticityNeurological defectsAggressive behaviorSelf-mutilationBiosynthesis of PyrimidinesBiosynthesis of PyrimidinesPyrimidine rings are synthesized independent of the ribose and transferred to the PRPP(ribose)Generated as UMP(uridine 5-monophosphate
9、)Synthesized from:Glutamine CO2 Aspartic acid Requires ATPNCCCHNCOCH3HOHNCCCNCHOHHNH2UracilCytosineRegulation of Pyrimidine BiosynthesisRegulation of Pyrimidine BiosynthesisRegulation occurs at first step in the pathway(committed step)2ATP+CO2+Glutamine=carbamoyl phosphateInhibited by UTPIf you have
10、 lots of UTP around this means you wont make more that you dont needXHereditary Orotic AciduriaHereditary Orotic Aciduria Defect in de novo synthesis of pyrimidines Loss of functional UMP synthetase Gene located on chromosome III Characterized by excretion of orotic acid Results in severe anemia and
11、 growth retardation Extremely rare(15 cases worldwide)Treated by feeding UMPWhy does UMP Cure Why does UMP Cure Orotic Aciduria?Orotic Aciduria?Disease(-UMP)No UMP/excess orotateDisease(+UMP)Restore depleted UMPDownregulate pathway via feedback inhibition(Less orotate)CarbamoylPhosphateOrotateUMPSyn
12、thetaseXFeedbackInhibitionBiosynthesis:Purine vs PyrimidineBiosynthesis:Purine vs PyrimidineSynthesized on PRPPRegulated by GTP/ATPGenerates IMPRequires EnergySynthesized then added to PRPPRegulated by UTPGenerates UMP/CMPRequires EnergyBoth are very complicated multi-step process whichyour kindly p
13、rofessor does not expect you to know in detailPyrimidine SalvagePyrimidine SalvageCan also be salvaged by reactions with PRPP -Catalyzed by Pyrimidine phosphoribosyltransferaseNucleoside kinaseDegradation pathways are quite distinct for purines and pyrimidines,but salvage pathways are quite similarW
14、ait a minute:Wait a minute:So far weve only made So far weve only made GMP,AMP,and UMPGMP,AMP,and UMPBeyond AMP,GMP and UMPBeyond AMP,GMP and UMPPurine BiosynthesisPyrimidine BiosynthesisBut other forms of these nucleotides are needed Two ProblemsTwo Problems These are monophosphates(i.e.GMP)-we nee
15、d triphosphates(i.e.GTP)for both DNA and RNA synthesis These are ribonucleotides-thats fine for RNA but we also need to make DNASynthesis of ribonucleotides first supports the RNA world theorySynthesis of UTP/CTP Synthesis of UTP/CTP(Easy Problem)(Easy Problem)Nucleotide DiphosphokinaseATPATPNCCCNCH
16、OHHNH2NCCCHNCOCH3HOHNCCCHNCOCH3HOHATP+Glutamine$So far weve made GTP,ATP,and UTP for incorporation into RNA$Also dGTP and dATP for incorporation into DNA$We still need dCTP for both RNA and DNA$We also need to generate dTTP for DNASpecific Kinases Convert NMP to NDPSpecific Kinases Convert NMP to ND
17、PMonophosphate kinases are specific for the basesNucleosideMonophosphatesNucleosideDiphosphatesMonophosphateKinasesAMP+ATP 2ADPGMP+ATP GDP+ADPAdenylate KinaseGuanylate KinaseConversion of Ribonucleotides to Conversion of Ribonucleotides to DeoxyribonucleotidesDeoxyribonucleotidesOHHHOHHHOCH2OHOH1 2
18、3 4 5 OHHHOHHHOCH2OHH1 2 3 4 5 BASEBASEDeoxyribonucleosideRibonucleosideSomehow we need to get rid of this damn oxygenRibonucleotideReductase$Catalyzes conversion of NDP to dNDP$Highly regulated enzyme$Regulates the level of cellular dNTPs$Activated prior to DNA synthesis$Controlled by feedback inhi
19、bitionactivityspecificityCell Cycle late G1Allosteric regulationOverall activity:+ATP,-dATPSubstrate specificity:ATP stimulate CDP,UDP reduction(d)TTP stimulates GDP reduction(d)TTP inhibits CDP,UDP reductiondGTP stimulates ADP reduction,inhibits GDP,CDP,UDP reductionring of nucleoside diphosphatesO
20、nce dNDPs are generated by ribonucleotide reductase a general kinase can phosphorylate to make the dNTPsSo far weve made(d)GTP,(d)ATP,and (d)CTP What about TTP?Synthesis of TTPSynthesis of TTP(Hard Problem)(Hard Problem)Methyl group is provided by N5,N10-Methylene tetrahydrofolateDihyrofolate reduct
21、ase recharges the Dihydrofolate to N5,N10-Methylene tetrahydrofolateNCCCHNCOCH3HOHNCCCHNCOCH3HOHThymidylateSynthaseCH3Role of Folate in dTMP SynthesisRole of Folate in dTMP SynthesisDihydrofolateReductaseThymidylateSynthaseDihydrofolateN5,N10-Methylene tetrahydrofolateTetrahydrofolatePurine Degradat
22、ionPurine DegradationSequential removal of bits and piecesEnd product is uric acidUric acid is primate-specificOther species further metabolizeuric acidExcreted inUrineXanthineOxidaseExcess Uric Acid Causes GoutExcess Uric Acid Causes GoutPrimary gout(hyperuricemia)Inborn errors of metabolism that l
23、ead to overproduction of Uric AcidOveractive de novo synthesis pathwayLeads to deposits of Uric Acid in the jointsCauses acute arthritic joint inflammationOffal foods such as liver,kidneys,tripe,sweetbreads and tongueAvoid:XanthineOxidaseAllopurinolXImmunodeficiency Diseases Associated with Purine I
24、mmunodeficiency Diseases Associated with Purine DegradationDegradationDefect in adenosine deaminaseRemoves amine from adenosineSCID-severe combined immunodeficiency“Bubble Boy”DiseaseDefect in both B-cells and T-cells(Disease of Lymphocytes)Patients extremely susceptible to infection-hence the Bubbl
25、eLymphocyteTherapies for SCIDTherapies for SCID Can be diagnosed in infants through a simple blood test(white cell count)Bone marrow transplant for infants Familial donor Continued administration of adenosine deaminase(ADA-PEG)Gene therapy-repair defective gene in T-cells or blood stem cellsPyrimidi
26、ne DegradationPyrimidine DegradationPyrimindine rings can be fully degraded to soluble structures(Compare to purines that make uric acid)Degradation pathways are quite distinct for purines and pyrimidines,but salvage pathways are quite similar90%of purines are salvagedMost de novo synthesis in liver
27、,highly regulatedCross regulation of purine and pyrimidine nucleotide biosynthesis assures balanced levels of these metabolitesDisruption of salvage or catabolism leads to diseaseAntimetabolitesAntimetabolitesOften drugs that inhibit cell growth are used to combat cancerMany of these compounds are a
28、nalogues of purine and pyrimidine bases or nucleotidesMany of these drugs must be activated by cellular enzymes They affect nucleic acid synthesis and tumor cells tend to be more susceptible since they are dividing more rapidly6-Mercaptopurine(6-MP)6-Mercaptopurine(6-MP)Purine AnalogueUsed clinicall
29、y to combat childhood leukemiaSince 1963 cure rate has increased from 4%to greater than 80%PRPP+6-MP6-mercaptopurine ribonucleotideInhibitor of Committed Step inde novo Purine BiosynthesisThis reaction is more active in tumor cellsCytosine Arabinose(araC)Cytosine Arabinose(araC)Metabolized to cytosi
30、ne arabinose 5-triphosphate(araCTP)Analogue of CTPIncorporated into DNA and inhibits chain synthesisUsed extensively for acute leukemiasCytosine ArabinoseDiffers only in the sugarNCCCNCHOHNH2HOHHHHOOHHOCH2NCCCNCHOHNH2HOCH2OHOHHHOHCytosine RiboseAZT as an Anti-HIV AgentAZT as an Anti-HIV AgentAzido-3
31、-deoxythymidinePyrimidine AnalogueHIV is a retrovirusRNA genome that is reverse-transcribed to DNAViral polymerase is inhibited by AZTNCCCHNCOHOHHHHOHN3CH3HOCH2AntifolatesAntifolatesAntifolates interfere with formation of dihydrofolate which is required for:dTMP synthesisde novo purine biosynthesisT
32、hymidylateSynthaseDihydrofolateN5,N10-Methylene tetrahydrofolateTetrahydrofolateDihydrofolateReductaseXAntifolate Agents Mimic FolateAntifolate Agents Mimic FolateHydroxyureaHydroxyureaSpecifically inhibits ribonucleotide reductaseInhibits DNA synthesis without affecting RNA synthesis or other nucle
33、otide poolsCleared from the body rapidly so not used extensively in the clinicH2N CONHOHAnti-Viral TherapiesAnti-Viral TherapiesTarget virally infected cellsTake advantage of aspects of viral metabolism that differ from normal cellular metabolismHIV-Human Immunodeficiency VirusHSV-HerpesvirusThe BIG PictureThe BIG Picture GMP,AMP,UMP on.Generation of dTMP Common features of clinically relevant antimetabolites/antifolates Antiviral agents-how are they specific for the virally infected cells?
