Nucleotide Metabolism Flashcards

Question

What is IMP and its role in de novo purine synthesis?

Answer 1

IMP = Inosine monophosphate - Purine base intermediate - Branch point for AMP & GMP biosynthesis (feedback regulated)

Answer 2

AMP requires Aspartate & GTP → Adenylosuccinate → {release fumarate} → AMP GMP requires Glutamine & ATP (via XMP) IMP → {NAD+ → NADH, dehydrogenase, to add =O} → XMP → {Glutamine + ATP + H2O, GMP synthase change =O to -NH2} → GMP *The ratios of ATP and GTP available to catalyze these reactions regulate (feedback) the synthesis of AMP vs GMP

Answer 3

Diphosphates via base specific kinases (non-specific for ribose/deoxyribose) Adenylate kinase: (d)AMP + ATP ←→ (d)ADP + ADP Uridylate kinase: (d)UMP + ATP ←→ (d)UDP + ADP ∆G~0, thermodynamic equilibrium, depends on the concentration ratios → [ADP]/[AMP] = [ATP]/[ADP] ∆G ~ 0 => [ADP]*[AMP]/[AMP]*[ATP] = 1

Answer 4

Triphosphates via ubiquitous non-specific kinase → Nucleoside diphosphate kinase (d)NDP + (d)N'TP ←→ (d)NTP + (d)N'DP ∆G~0, thermodynamic equilibrium, depends on the concentration ratios → [UTP]/[UDP] = [ATP]/[ADP]

Answer 5

~ 7x more RNA than DNA ~ 7x more NTP that dNTP ~ 7x more NTP than NDP ~ 7x more NDP than NMP RNA/NTP > DNA/dNTP > NDP > NMP

Answer 6

1. Feedback inhibition by nucleotides (AMP/GMP and ADP/GDP → IMP branch point) 2. Feedback inhition by nucleotides → Steps 1 and 2 3. Feedforward by PRPP (for Step 2 which has PRPP as a substrate) 4. Inhibition & stimulatio for AMP and GMP syn *AMP, ADP, ATP are all nucleotides

Answer 7

- Most versatile enzyme cofactor known - Carries 1 carbon « unit » in 3 different oxidation states - Carries 1 carbon « unit » in 5 different chemical forms - From folic acid (found in leaves) → humans get it from their diet (eating salad)

Answer 8

Antibiotics Bacteria synthesize folate de novo from p-aminobenzoic acid while human get it from food (salad) Due to their similarity to p-aminobenzoic acid (the central section of THF)→ they inhibit synthesis of folate *Since some bacterias synthesize their own folate, these drugs inhibit the synthesis → imapirs capcity to synthesize nucleotides → death

Answer 9

*THF is a Carbon carrier, can carry carbon under 3 forms Methanol → most reduced (3x H) → carries Methyl (-CH3) → Derivative = N5-Methyl-THF Formaldehyde → (2x Hs) → carries Methylene (-CH2-) → Derivative = N5, N10-Methylene-THF Formate → most oxidized (1H) → 3 different carried groups a) Formyl (-CH=O) → N5-Formyl-THF,N10-formyl-THF b) Formimino (-CH=NH) → N5-Formimino-THF c) Methenyl (-CH=) → N5,N10-Methenyl-THF

Answer 10

1. Methionine cycle (5-Methyl-THF) 2. Thymidylate synthase (N5,N10-methylene-THF) 3. Purine de novo (N10-Formyl-THF)

Answer 11

Pregnant women → prevent neural tube defects in embryos

Answer 12

1. Removing the 2'OH makes the DNA phosphodiester backbone more stable → 2'OH attacks the phophate from backbone 2. The base thyine allows spontaneuous deamination of cytosine (→ Uracil) to be detected) because uracil is not a possible base of DNA

Answer 13

Adenine → Hypoxanthine Guanine → Xanthine Cytosine → Uracil

Answer 14

DNA is more reduced Carried at the level of DNPs (diphosphates) by the enzyme: ribonucleotide reductases Reductase because the 2' O is removed → add an H (reduction)

Answer 15

NADPH → FADH2 → HS-SH → HS-SH → dNDP 1. Thioredoxin reductase 2. Thioredoxin 3. Ribonucleotide reductase Pass through thiols (cysteine residues) of thioredoxin reductase to thioredoxin and to finally RR

Answer 16

Reducing power → NADPH *NADPH/NADP+ = 100 Oxidizing power → NAD+ NAD+/NADH = 1000

Answer 17

Heterodimer (dimer of homodimers) 2 Allosteric sites on each regulatory subunit: - Activity controlled by ATP/dATP levels - Specificity controlled by dATP/dGTP/dTTP *2 types of feedback regulation to maintain equilibrated levels of dNTPs Catalytic subunit: Stable enzyme has a tyrosine free radical - Inhibited by hydroxyurea (converts tyrosine radical → tyrosine) Bacteria often encode their own RR as they depend on it for DNA synthesis → target for many antibiotics or cancer treatments Other types of RR exist with B12, Fe/S or other cofactors than tyrosine

Answer 18

Base excision repair 1. Uraci-DNA glycosylase cleave Uracil base → apurinic site 2. Apurinic/Apyrimidinic (AP) endonuclease cleaves DNA backbone 3. Resynthesis from complementary strand *Excessive repair causes mutations

Answer 19

A specific enzyme UTPase hydrolyzes dUTP → dUMP to decrease its concentration and prevent its incorporation *Incorporation of dUTP mimics deamination of cytosine, which is recognized by DNA repair mechanisms

Answer 20

Start with Aspartate → Uridylate (UMP) → UTP A) UMP → UTP → CTP → RNA B) UMP → UTP → CTP → CDP →dCDP → dCTP → DNA C) UMP → UDP → dUDP → dUTP → dUMP → dTMP → dTTP → DNA

Answer 21

C-C-C-N → Aspartate N → Glutamine amide C → HCO3-

Answer 22

Pyrimidine ring is assembled separately, phosphoribose (PRPP) added at the end to make nucleotide Purine ring is assembled from the Nitrogen which is first added to PRPP (assembled on the ribotide)

Answer 23

1. 2ATP + Glutamine + HCO3- + H2O → Carbamoyl phosphate + 2ADP + Glutamate + Pi Enzyme = Carbamoyl phosphate synthetase 2 enzymes in animals: - In mitochondria for urea cycle - In cytosole for pyrimidine synthesis *Allows step 1 regulation in animals Bacteria have only 1 enzymes for both pathways so regulation of the pyrimidine synthesis is done at step 2

Answer 24

*Step 2 of pyrimidine de novo synthesis Well-studied example of allosteric regulation - Activated by ATP - Inhibited by CTP → Affects the Km of the enzyme (affinity)

Answer 25

2. Carbomoyl phosphate + Aspartate → Carbamoyl aspartate + Pi Enzyme = Aspartate transcarbamoylase (ATCase) *All atoms are there + COO- group because charged groups prevent the ring from diffusing out of the cell *Bacteria regulate this step

Answer 26

Channeling between Steps 1, 2 and 3 Intermediate goes to the mitochondrial membrane for step 4 Channeling between steps 5 and 6 Allows to increase throughput, decrease cellular concentrations of intermediates Channeling is done by mulitple enzymes assembling in complexes *Channeling only occurs in eukaryotes because bacteria needs enzyme 1 to be isolated because it sevres the urea cycle as well

Answer 27

Leads orotic aciduria - Caused by build up in Orotate

Answer 28

Step 3 → Close the pyrimidine ring Carbamoyl aspartate → {Dihydroorotase} → Dihydroorotate + H2O *Dehydration Step 4 → Oxidation (makes double bond) Dihydroorotate + Quinone → {dihydroorotate dehydrogenase} → Reduced quinone + Orotate *Occurs in the mitochondrial membrane → 2 electrons → CoQ

Answer 29

Step 5 → put Orotate onto the ribotide Orotate + PRPP → {orotate phosphoribosyl transferase} → PPi + OMP *OMP = UMP + COO- Step 6 → Removal of COO- to make UMP OMP → {OMP decarboxylase} → UMP + CO2

Answer 30

Orotate = Uracil + COO- *The COO- which prevents the base to diffuse out of the cell

Answer 31

*At level of the triphosphates - N from glutamine UTP + Glutamine + ATP + H2O → {CTP synthetase} → CTP + Glutamate + ADP + Pi *From UMP, → specific kinase → UDP → non-specific kinase → UTP

Answer 32

Animals → regulation at step 1 Bacteria → regulation at step 2 (ATCase) Inhibition by pyrimidine nucleosides triphosphates (feedback) Activation by ATP and PRPP (feedforward) *ATCase → activated by ATP and inhibition CTP

Answer 33

1. The direction of the enzyme reactions depends on the concentration of reactants 2. [NADPH] > [NADP+] while [NADH] < [DNA+] 3. Base-specific nucleotide kinases determine which nucleotide becomes diphosphates 4. NADPH drives thioredoxin reduction

Answer 34

ATP activates and CTP inhibits ATCase in an allosteric manner This happens at LOW aspartate concentrations (al high aspartate concentrations, the enzyme is saturated no matter ATP or CTP concentrations) *2nd step of pyrimidine de novo synthesis

Answer 35

UMP → UDP → UTP → CTP → CDP → CMP - Consumes 1 ATP for the amination of UTP → CTP - The other ATPs are regained back on the other side Advantage: The cell has much higher levels of triphosphates that monophosphates (~50X) so the Km of the enzyme can much higher (no need for such high affinity)

Answer 36

Formation of dUTP: - Ribonucleotide reductase (at level of diphosphates) is not specific for the base - Uridylate kinase (UMP/dUMP → UDP/dUDP) is not specific for ribose vs deoxyribose → nucleoside diphosphate kiase (non-specific for anything) - dUTP can also come from deamination of dCTP Solution: 1. dUTPase degrades dUTP → dUMP 2. dUMP is then converted to dTMP by thymidylate synthase (level of monophosphates)

Answer 37

It converts dUMP → dTMP - Adds a methyl which comes from N5,N10-methylene tetrahydrofolate - Not expected because methylene has 2Hs and has to give a methyl in its most reduced for (CH3) to Uracil → methylene is reduced to methyl in exchange with oxidation of THF → DHF (dyhydrofolate) - Important target for drugs against cancer and bacteria → essential for thymine synthesis (DNA) - Required for DNA synthesis, but not RNA - Synthesis at the level of monophosphates (dUMP coming from dUTP which we don't want)

Answer 38

1. Fluorodeoxyuridylate (FdUMP): dUMP the normal substrate + fluor group where the methyl must be added - Competitive inhibition (binds in the substrate binding site) - Suicide inhibitor → binds to the enzyme and "kills" its activity 2. Methotrexate, Aminopterin, Trimethoprim inhibits DHF → THF (by DHF reductase) *Electron donor = NADPH - THF analogs - Competitive inhibitors - Anti-cancer and antibacterial by preventing them to synthesize DNA

Answer 39

Dihydrofolate reductase - Thymidylate synthesis requires reduction of the methylene → methyl to add it to the uracil and make thymine - This requires oxidation of THF → DHF - It then has to be reduced back the THF → called TS cycle - NADPH = electron source In the next step, A new methylene group is added to THF from serine Cycle = Thymidylate synthase (N5,N10-Methylene-THF → DHF) → Dihydrofolate reductase (DHF → THF) → Serine hydroxymethyl transferase (THF → N5,N10-methylene-THF)

Answer 40

Conversion of base → to ribonucleotide: Phosphooryibosyl transferase PRPP + base X → rNMP + PPi

Answer 41

Conversion of base → ribonucleotide (Phosphoribosyl transferase) Interconversion of bases a nucleosides (Nucleoside phosphorylase) Conversion of nucleoside → nucleotide (nucleoside kinase) Base exchange into deoxynucleosides (nucleoside transglycosylase) Interconversion by base alterations (deaminases) Reutilization of nucleotides (nucleoside monophosphate kinase & nucleoside diphosphate kinase)*seen as part of de novo pathway

Answer 42

PRPP + Purine → Purine ribonucleotide + PPi BASE → NUCLEOTIDE (jump 2 levels, Slavage pathway) The Purine can be Hypoxanthine (→ Inosinate) or Guanine (→ Guanylate) *genetic loss of HGPRT activity causes Lesch-Nyhan disease *Other enzymes for adenine + uracil (purines, but not this enzyme)

Answer 43

It is a genetic disease caused by the loss of Hypoxanthine/Guanine phosphoribosyl transferase (HGPRT) Symptoms: gout and mental retardation

Answer 44

- Nucleoside phosphorylase - Nucleoside transglycosylase (exchange bases) *These enzyme are substrate-specific to avoid making thymine ribonucleotides

Answer 45

Ribonucleoside or deoxyribonucleoside (beta anomer) → Ribose 1-P or Deoxyribose 1-P (alpha anomer, until add another base by nucleoside phosphorylase) *Not 5’ phosphate, but ribose + beta-base at 1’C

Answer 46

Switch from 1 base to another on a ribose Deoxyadenosine → (Deoxyribose 1-P alpha anomer) → Deoxythymidine *Nucleoside phosphorylase can catalyze the subreactions

Answer 47

1. Mutase (Ribose 5-P ←→ Ribose 1-P) 2. Deaminase 3. Phosphorylase 4. Transglcycosylase 5. Kinase (adds 5’ phosphate ro ribose) 6. Transferase 7. PRPP synthetase (ribose-5P → ribose-5P-1PPi, consumes 1 ATP)

Answer 48

Funel downwards: nucleotide (monophosphate) → nuceloside → base (Hypoxanthine, Guanine) → Xanthine → uric acid

Answer 49

1. AMP → {AMP deaminase} → IMP → {nucelotidase} → Inosine → {purine nucleoside phosphorylase, release Ribose 1-P) → Hypoxanthine → {xanthine oxidase} → Xanthine → {xanthine oxidase} → Uric acid 2. AMP → {nuleotidase} → Adenosine → Inosine → same *Can deaminate at the level of AMP or at level of the bases

Answer 50

Genetic disease SCID (severe combined immunodeficiencies)

Answer 51

Xathine oxidase → by inhibiting it, we prevent build up of uric acid through purine degradation

Answer 52

Catalyze nucleotides → nucleosides by hydrolysis of the phosphate

Answer 53

Depositions of uric acid in joints - Painful, prevalent in males, middle ages - Thought to be due to excess meat - More likely kidney dysfunction Symptomatic of metabolic imbalance → exccess de novo purine synthesis (can be due to high PRPP levels which are feedforward activators)

Answer 54

How is it important for pigeons? - It if the final degradation product of purines - Allows elimination of nitrogen in birds → with conservation of water *Purine de novo pathway discovered by studying pigeons Uric acid is not soluble when protonated (at low pH) → birds lower their pH → uric acid precipitates → white paste is excreted

Answer 55

- Uric acid is secreted by birds and primates - Marine invertabrates secrete NH4+ → cheapest way (ATPwise), but requires a lot fo water - Elimination costs a lot of water - To not use too much water, birds keep lower pH → uric acid precipitates when protonated

Answer 56

Funnel downward: Nucleotide → nucleoside → base → uracil (Thymine) 1. Deamidate to carbonyl 2. reduce ring CMP → {nucleotidase} → Cytidine → {Cytidine deaminase, release NH4+} → Uridine → {Uridine phosphorylase} → Uracil → {dihydrouracil dehydrogenase} → Dihydrouracil UMP (dTMP) → {nucleotidase} → uridine (deoxythymidine) → Uracil → {dihydrouracil dehydrogenase} → Dihydrouracil *Phosphorylase dissociates the ribose from the base (base specific)

Answer 57

1. They are structural analogues of PABA (part of THF) 2. They do not affect human cells because we don’t do synthesis of folate, we get it from diet

Nucleotide Metabolism Flashcards

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