Nucleotides metabolism

8.1 Purine metabolism -8.1.1 The Biosynthesis of Purines -8.1.2 Purine Salvage -8.1.3 De-oxyribonucleotide Synthesis -8.1.4 Purine Degradation8.2 Pyrimidine metabolism -8.2.1 Biosynthesis of Pyrimidines -8.2.2 Pyrimidine DegradationOutline

Biological Roles of NucleotidesMonomeric units of nucleic acids * Energy currency(ATP) *Regulation of physiological processesAdenosine controls coronary() blood flowcAMP and cGMP serve as signaling moleculesPrecursor function (GTP to tetrahydrobiopternin)Coenzyme components ( 5-AMP in FAD/NAD+)Activated intermediates: UDP-GlucoseAllosteric effectors- regulate themselves and others

Deoxyribonucleotide umolfoodprotein nuclear acid (RNA and DNA) (intestine)Endonucleases(phosphodiesterase) mononucleotideNucleotidase(phosphoesterase) nucleosidePhosphatenucleosidaseRibose or ribose-1-phosphatebaseNuclear acid digestionUric acid (purines)-ureidopropionate( primidines)RNaseDNase(stomach)ribonucleotide mmol()excrete

8.1.1 Nucleotide BiosynthesisFor both purines and pyrimidines there are two means of synthesis - de novo (from bits and parts) - salvage (recycle from pre-existing nucleosides,and bases) Ribose generates energy, but purine and pyrimidine rings do not Nucleotide synthesis pathways are good targets for anti-cancer/antibacterial strategies

Bases/Nucleosides/Nucleotides BaseBase + Sugar=NucleosideBase + Sugar + Phosphate=NucleotideAdenineDeoxyadenosineDeoxyadenosine 5-triphosphate(dATP)

The Pyrimidine RingThe Purine Ring

PurineOH2N26AG

PyrimidineCytosineThymine

De novo purine biosynthesisJohn Buchanan (1948) "traced" the sources of all nine atoms of purine ring1. In de novo synthesis, Inosine-5'-P (Inosine Monophosphate, IMP) is the first nucleotide formed2. It is ,then, converted to either AMP or GMP Location: liver cellular Cytoplasm De novo purinenucleotide synthesis proceeds by the synthesis of the purine base upon the ribose sugar moiety

The metabolic origin of the nine atoms in the purine ring systemN-1: aspartic acid C-2:THF - one carbon unitsN-3: glutamine C-4, C-5, N-7: glycine C-6: CO2 C-8: THF - one carbon unitsN-9: glutamineN-1C-2N-3C-6C-8, ,, CO2

1. First, synthesis Inosine-5'-P (Inosine Monophosphate, IMP)

R-5'-PPRPP synthetasePP-1'-R-5'-P(PRPP)

5-,PRA T1/2 30s

(FGAR)

(FGAM)

5-(AIR)

5--4-(N-)-(SAICAR)

5--4-(AICAR)

5--4-(FAICAR)

NH3 via aspartyl- succinatePRPPInosine monophosphate

2.Second, Making AMP and GMP

ADPADPATPATPADPAMPATPGDPADPGTPATPADPGMPATP

Regulation of De Novo Synthesis

ATP provides the energy for GMP synthesisGTP provides the energy for AMP synthesis3. cross regulation occurs from IMP to AMP and GMP2.End product inhibition and feed forward regulation(A bunch of steps you dont need to know)Purines are synthesized on the Ribose ringFeedbackInhibition

Committed Step

8.1.2 Salvage Pathway for PurinesSalvage pathways are particularly important in brain/marrow that lack de novo purine synthesis

Lesch-Nyhan Syndrome(-)Absence of HGPRTaseX-linked (Gene on X)Occurs primarily in malesCharacterized by:purine synthesis is increased 200-foldIncreased uric acidSpasticity()Neurological defectsAggressive behaviorSelf-mutilation()

XMP AMPSIMPInter-conversion of Purine nucleotidesAMPGMP

8.1.3 Deoxyribonucleotide BiosynthesisDeoxyribonucleosideRibonucleosideRibonucleotideReductase

ADPdADPGDPdGDPUDPdUDPCDPdCDPTDPdTDP

Deoxyribonucleotide Biosynthesis ?Mg2+ Ribonucleotides can be converted to deoxyribonucleotides by Ribonucleotide Reductase at the diphosphate level

The ribonucleotide reductase, An (R1)2(R2)2- type enzyme , has R1 (86 kD) and R2 (43.5 kD) two subunits E. coli Ribonucleotide Reductase Regulates the level of cellular dNTPs

dCDP+ATPdCTP+ADPdUDP+ATPdUTP+ADPdGDP+ATPdGTP+ADPdADP+ATPdATP +ADPdTTP?dNDPdNMP+Pi

Regulation of dNTP SynthesisThe overall activity of ribonucleotide reductase must be regulated Balance of the four deoxynucleotides must be controlled ATP activates, dATP inhibits at the overall activity site ATP, dATP, dTTP and dGTP bind at the specificity site to regulate the selection of substrates and the products made

Regulation of dNTP Synthesis

over-growth + Heterogeneity ( nucleotides + protein )Tumor How to inhibit the biosynthesis of the tumor cells?for anti-cancer strategies(antibacterial)

Chemotherapeutic Agents1. Analogs of purine:8-6-inosine(8-azoguanine)(6-mercaptoguanine)

2. Analogs of amino acids:Glnazaserine6--5-(diazonnorleucine)Inhibit the reactions of the Gln

3. Analogs of Folic acidR=Haminopterin,R=CH3methotrexate,,MTX,FH4

PRPPGln6MP(azaserine)PRAGARFGARFGAMMTXazaserineAICARMTXFAICARIMPAMPGMPPRPPPPiPPiPRPP6MP6MP6MPazaserineAIGPRPPPPiThe mechanism of the Chemotherapeutic Agents

AMPIGMPGXXO XOExcreted inUrineSequential removal of bits and piecesEnd product is uric acidXO: Xanthine Oxidase 8.1.4 Purine catabolism

The scale of uric acid (normal value) : 0.120.36mmol/L; male, 0.27mmol/L; formale, 0.21mmol/L0.48mmol/L(8mg%),Xanthine Oxidase and Gout

IAllopurinol, which inhibits XO, is a treatment of gout

IallopurinolPRPPAllopurinol nucleotideXOPurine nucleotidesThe mechanism of allopurinol as a treatment of goutUric acidsX

8.2 Pyrimidine BiosynthesisPyrimidine Biosynthesis: In contrast to purines, First, synthesis of the pyrimidine ring; Next, attachment of ribose-phosphate to the ring

De Novo Pyrimidine BiosynthesisThe metabolic origin of the six atoms of the pyrimidine ring

CTP From UTP at the triphosphate levelUDPADPUTPATPADPUMPATP

1. Thymine nucleotides are made from dUMP, which derives from dUDP, dCDP2. Biosynthesis of deoxyribonucleotides by ribonucleotide reductase3. Biosynthesis of thymidine monophosphate (dTMP) by thymidylate synthaseSynthesis of Thymine Nucleotides

dUMPFH2N5,N10- methylene FH4FH4NADPH+H+NADP+dTMPdTDPADPdTTPATPADPdTMPATPThymidylate synthase methylates dUMP at 5-position to make dTMPN5,N10-methylene THF is 1-C donor

Regulation of Pyrimidine Synthesisde novoAspartate transcarbamoylase (ATCase ) catalyzes the condensation of carbamoyl phosphate with aspartate to form carbamoyl-aspartate Note that carbamoyl phosphate represents an activated carbamoyl group Feedback Inhibition

Regulation of Pyrimidines BiosynthesisRegulation occurs at first step in the pathway (committed step)2ATP + CO2 + Glutamine = carbamoyl phosphate

CPS IICarbamoyl phosphate for pyrimidine synthesis is made by carbamoyl phosphate synthetase II (CPS II ) This is a cytosolic enzyme (whereas CPS I is mitochondrial and used for the urea cycle) Substrates are HCO3-, glutamine, 2 ATP

Allosteric regulation of pyrimidine biosynthesis

CPS-I vs. CPS-II ?

Biosynthesis: Purine vs. Pyrimidinestart with ribose, build on nitrogen baseRegulated by GTP/ATPGenerates IMPRequires Energybuild nitrogen base then added to PRPPSynthesized Regulated by UTPGenerates UMP/CMPRequires EnergyBoth are very complicated multi-step process whichyour kindly professor does not expect you to know in detail

Salvaging PyrimidinesPyrimidines+PRPP Nucleoside+PPi ()A second type of salvage pathway involves two steps and is the major pathway for the pyrimidines, uracil and thymine Base + Ribose 1-phosphate = Nucleoside + Pi (nucleoside phosphorylase) Nucleoside + ATP Nucleotide + ADP (nucleoside kinase-irreversible)

Analogs of pymidines /pymidine nucleosides:5-5-Fu Cytarabine Cyclocytidine Inhibitors of pymidines synthesis are cancer drugs

UMPUDPUTPdUDPdUMPdTMPCTPCDPdCDPMTX5FU(5FdUMP/5FUTP)Cytarabine azaserine

NH3NADP+H2OCO2+NH3H2N-CH2-CH2-COOH-AlanineCUPyrimidine Catabolism-1

NADP+CO2+NH3-aminoisobutyrate-H2OTDHTPyrimidine Catabolism-2-

overview5'-P-RPRPPIMPdAMPGMPdGMPAMPdADPGDPdGDPADPdATPGTPdGTPATPUMPCMPdUMPUDPCDPdUDPUTPCTPdUTPdTMPdCMPdTDPdCDPdTTPdCTPCO2+GlnH2N-CO-POMPDe novo synthesisdUDP

CPS-I vs. CPS-IIIII() N-UMP (CPS-I)(CPS-II)

? A. B. C. D. E.

2. 5-A. DNAB. C. D. E.

3. A. B. C. D. E.

4. A. B. 6-C. 1-D. 1,6-E. 5-

5. A. B. C. D. E.

6. , ?A. B. C. D. E.

7. -CMPAMPTMPUMPIMP

, ?GTP-

9. PRPP, A. R-5-PPRPPB. C. PRPPD. IMPAMPE. IMPGMP

10. A. 5-B. FH4C. D. E.

11. The main tissue of de nove synthesis of purine nucleotide in vivo isA. thymus glandB. villous coat of small intestineC. liverD. spleenE. marrow

12. The main end product of purine nucleotide katabolic metabolism in human body isA. ureaB. creatineC. CreatinineD. uric acidE. -alanine

13. The methyl of thymine come fromN10-CHO FH4N5,N10=CH-FH4N5,N10-CH2-FH4N5-CH3FH4N5-CH=NHFH4

14. 6-mercapto-purine nucleotide doesnt suppressA. IMPAMPB. IMPGMPC. PRPP amide transferaseD. Purine phosphoribosyltransferaseE. Pyrimidine phosphoribosyltransferase

15. A B CO2C D E

16. PRPPA B C D NMPNDPNTP

17. A B C D

18. A B C

19. The compound which can produce feedback suppression of purine nucleotide synthesis is A IMPB AMPC GMPD uric acid

20. Which compound produce uric acid as its decomposed metabolism end product ?A AMPB UMPC IMPD TMP

Figure 22-31-01Figure 22-31-02Figure 22-31-03Figure 22-31-04Figure 22-31-05Figure 22-31-06Figure 22-31-07Figure 22-31-08Figure 22-31-09Figure 22-31-10Figure 22-31-11A-PRT(phosphoribosyltransferases) is not very important because we generate very little adenine. (the catabolism of adenine nucleotides and nucleosides is through inosine) HG-PRT, though, is exceptionally important and it is inhibited by both IMP and GMP. This enzyme salvages guanine directly and adenine indirectly. Remember that AMP is generated primarily from IMP, not from free adenine PRTs catalyze the addition of ribose 5-phosphate to the base from PRPP to yield a nucleotideBase + PRPP = Base-ribose-phosphate + PPiHighly regulated enzymeActivated prior to DNA synthesisControlled by feedback inhibition by dATP, and complex positive regulation by TTP, dGTP and dGTP

Figure 22-40XO in liver, intestines (and milk) can oxidize hypoxanthine (twice) to uric acid Humans and other primates() excrete uric acid in the urine, but most N goes out as urea Gout occurs from accumulation of uric acid crystals in the extremities

Figure 22-41Uridine + ATP UMP + ADP; uridine kinaseNucleoside phosphorylase/kinase: U,C,T + ribose 1-P nucleosides + PiThymine + deoxyribose-1-P thymidine + Pi; thymine phosphorylase Thymine + ATP dTMP + ADP; thymidine kinaseThymidine kinase activity changes during cell cycle; very active during DNA synthesis and is inhibited by dTTP

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Nucleotides metabolism