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Gastrointestinal System > Nucleotide Metabolism - Roth 3/14/16 > Flashcards

Nucleotide Metabolism - Roth 3/14/16 Flashcards

1
Q

roles of nucleotides in the body

A
  1. building blocks of nucleic acids (DNA, RNA)
  2. energy currency in the cell (ATP, GTP)
  3. carriers of activated intermeds (UDP-glucose, SAM)
  4. structural components of essential cofactors (NAD+)
  5. metabolic regulation, signal molecules (cyclic AMP)
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2
Q

nucleotide nomenclature

A

base (AGCU, T)

nucleoside: base + sugar (adenosine, guanosine, cytidine, uridine, thymidine)

nucleotide: base + sugar + P (_TP, _DP, _MP)

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3
Q

sources of nucleotides

A
  1. de novo synthesis
    * base built from scratch
  2. salvage pathways
  • reuse “preformed bases” to build nts (obtained from nucleic acid breakdown in cells or dietary nucleic acids)
  • used often in
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4
Q

nucleotide degradation overview

A

mononucleotides are degraded → nucleosides (ultimately to ribose-1P) + free bases

free base either salvaged to form mononucleotides or shuttled to degradation

purine bases (A, G) degraded to uric acid

pyrimidine bases degraded to soluble pdts

  • beta-aminoisobutyrate (T)
  • beta-ureidopropionate (C, U)
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5
Q

de novo synthesis of purine nts

  1. synthesis of sugar-P backbone
A

sugar-P backbone (aka PRPP)

ribose-5P + ATP → PRPP

  • PRPP synthetase
    • ​+ : inorganic P
      • : purine ribonucleotides (ADP, ATP, GDP, GTP)
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6
Q

de novo synthesis of purine nts

  1. committing PRPP to purine synthesis
A

committed step for making purines

PRPP + Gln → PRA (5-phosphoribosylamine)

  • PRPP-amino-transferase
    • ​+ : PRPP
      • : AMP, GMP, IMP (parent molecule for AMP/GMP)
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7
Q

glutamine analogs

A

clinically, antibiotics azaserine and DON are Gln analogs

  • irreversibly inhibit PRPP-amin-transferase → prevent committed step of purine synth!
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8
Q

synthesis of IMP

A

IMP is the parent structure for AMP, GMP

PRPP + Gln → IMP

  • need energy (ATP)
  • carbon sources (THF, CO2)
  • amino acid (Gln, Asp, Gly)
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9
Q

importance of THF to purine synthesis

role of methotrexate and aminopterin

A

required for purine synthesis

  • humans cant synthesize it, must get it in diet

folate → DHF → THF [2x DHFR enzyme action]

  • methotrexate and aminopterin are chemo agents that competitively inhibit DHFR and halt purine synth in humans → cell death in rapidly dividing cells (not selective just for cancer)
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10
Q

importance of THF to purine synthesis

role of sulfonamides

A

bacteria can synthesize folate

PABA is the precursor for THF in bacteria

folate → DHF → THF [2x DHFR enzyme action]

  • sulfonamides are structural analogs of PABA that competitively inhibits folic acid synth in bacteria
  • trimethoprim binds more tightly to bacterial DHFR than mammalian → effective antimicrobial agent
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11
Q

purine synthesis

IMP → AMP

A

IMP + GTP (energy) + Asp (N source) → adenylosuccinate

adenylosuccinate → fumarate + AMP

*GTP is needed to synthesis AMP

*AMP demonstrates feedback inhibition

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12
Q

purine synthesis

IMP → GMP

A

IMP + NAD+ → XMP

  • IMP DH (inhibited by ribavirin, high GMP)

XMP + ATP (egy) + Gln (N source) → GMP

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13
Q

inhibitors of IMP dehydrogenase

A

ribavirin

  • antiviral used to treat HepC
  • inhibits IMP dehydrogenase → depletes intracellular pools of GMP

mycophenolic acid

  • blocks nt synth in T and B cells → prevents organ rejection
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14
Q

regulation of purine biosynthesis:

what determines whether IMP → GMP or AMP?

reciprocity

A

feedback inhibition

  • high GMP : IMP → AMP
  • high AMP : IMP → GMP

reciprocity: ATP req for GMP synth; GTP req for AMP synth

  • high AMP/GMP indicates high ATP/GTP, so favors the synth of the other
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15
Q

adding phosphates

conversion of NMP → NDP → NTP

A

nucleoside monophosphate kinases add P group to NMP

  • each base has its own NMP kinase

nucleoside diphosphate kinase adds P group to NDP

  • same NDP kinase acts on all bases
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16
Q

point of reciprocity in purine synthesis

A

ATP and GTP synth are individually regulated to control total level of purines and relative amts of A and G

reciprocity rules: ATP powers synth of GMP, GTP powers synth of AMP

17
Q

purine salvage pathway

A

recycling free purine bases from hydrolytic degradation

PRPP + base → purine ribonucleotide

  • much more energy efficient than de novo synth
  • key in tissues with low de novo synth (ex. brain)
  • ribose-P comes from PRPP
18
Q

two salvage pathways (and key enzymes)

A

1. APRT (adenine phosphoribosyl transferase)

  • adenine + PRPP → AMP

2. HGPRT (hypoxanthine-guanine phosphoribosyl transferase)

  • guanine + PRPP → GMP
  • hypoxanthine + PRPP → IMP
19
Q

what happens when salvage pathways are blocked?

A

de novo synthesis is turned on!

water/tap/drain? look at slide

20
Q

Lesch-Nyhan syndrome

A

X linked recessive :

HGPRT deficiency → reduced IMP and GMP salvage

symptoms: spasticity, mental retardation, aggression, self-mutilation, gout

pathophysio:

  • insufficient GTP during brain devpt (brain more reliant on salvage pathway than other tissues)
  • GTP is involved with dopaminergic neuron diff and dopamine biosynth
    • fewer dopaminergic neurons
    • less dopamine synth

HGPRT deficiency → hypoxanthine, guanine, PRPP buildup

  • activates de novo synth → more hypoxanthine and guanine made that you cant do anything with → uric acid buildup → gout
21
Q

degradation of purines to uric acid

A
  1. phosphate is removed from AMP, GMP, or IMP → adenosine, guanosine, inosine [nucleotidase]
    * side rxn that can happen…AMP → IMP [AMP deaminase, removes an amino group]
  2. removal of amino group from adenosine → inosine [adenosine deaminase]
  3. removal of ribose from inosine and/or guanosine [purine nucleoside phosphorylase, PNP]
  • inosine → hypoxanthine
  • guanosine → guanine
  1. hypoxanthine, guanine → xanthine → uric acid
  • 2x xanthine oxidase
  • uric acid can be an antioxidant, excreted in urine
22
Q

adenosine deaminase deficiency

→

severe combined immunodeficiency (SCID)

A

ADA deficiency accounts for 15% of SCID cases

symptoms: severe bacterial/viral/opportunistic infections in early life → can be fatal

pathophys: severe deficit of B and T lymphocytes

tx: bone marrow transplant with or without gene therapy/ERT

  • prophylactic IgG
23
Q

adenosine deaminase deficiency

A

leads to accumulation of DATP (50x higher conc than normal)

potential explanations for effects on B and T cells…

  1. high [DATP] shuts down ribonucleotide reductase, stops dNTP synth/DNA synth
  2. high [deoxyadenosine] shuts down S-adenosylhomocysteine hydrolase - req for methylation of RNA/DNA bases
  3. high [adenosine] → high cAMP levels

*no suitable explanation for effects ltd only to B and T cells :(

24
Q

PNP deficiency

A

genetic deficiency of PNP

commonly presents in childhood

symptoms: recurrent bacterial/viral/opportunistic infections

pathophys: severe deficit of T lymphocytes

tx: bone marrow transplant

25
Q

uric acid

gout

A

uric acid is close to solubility limit in serum

pH and temp can affect solubility

buildup of uric acid → deposition of monosodium urate crystals in tissues: GOUT

primary hyperuricemia: error in uric acid metab

  • overactive PRPP synthetase
  • dereg of PRPP-amino-transferase
  • Lesch Nyhan syndrome

secondary hyperuricemia

  • malignancy (elevated cell turnover)
  • chronic renal insufficiency (underexcretion - most common cause of gout)
  • G6Pase def (von Gierke disease; increased G6P stimulates PRPP production)
  • meds like HCTZ

symptoms: joint pain, swelling, warmth, redness, tenderness in joints

tx: allopurinol : analog of hypoxanthine, inhibits xanthine oxidase

  • competitive inhibitor of xanthine oxidase
  • xanthine oxidase converts it into alloxanthine : potentnt irreversible inhibitor of enzyme
  • buildup of hypoxanthine → salvaged to produce IMP
    • IMP is an inhibitor of PRPP-amino-transferase (de novo purine synth) → less purine synth, less degradation!

febuxostat, non-purine analog that also inhibits xanthine oxidase

26
Q

xanthine and hypoxanthine are ore soluble than uric acid and can be excreted

why bother making uric acid in the first place?

A

urate in serum is also an antioxidant → scavenges ROS

27
Q

purine nucleotide cycle

  • anaplerotic rxn
  • where is this rxn taking place
A

purine nucleotide cycle links nucleotide metabolism to TCA cycle via fumarate (generated during IMP → adenylosuccinate → AMP + fumarate → IMP)

  • indirectly replenishes an intermediate of the TCA cycle = anaplerotic rxn

during sustained muscle activity, TCA cycle intermeds need to be replenished for energy production

  • in muscle cells, purine nt cycle can do this by replenishing fumarate; 3 enzymes involved are upreg’d in muscle cells
28
Q

de novo pyrimidine synthesis

A

remember: NOT SYNTHESIZING parent base ON the PRPP backgone (de novo purine synth)…

  • parent base synthesized entirely
  • parent base attached to PRPP

1. synthesizing carbamoyl phosphate (regulated step of pyrimidine synth)

2ATP + CO2 + Gln → carbamoyl phosphate

  • carbamoyl phosphate synthetase II (cytosolic CPS II; distinct from CPS I, mitochondrial rate limiting step in urea synth, protein metab!!!!)
    • : ATP, PRPP
    • : UTP

2. competing pyrimidine ring and ring closure

carbamoyl P (2 C) + 4 Asp → orotate (ringed, 6C)

  • rxn is coordinated by CAD - large polypep including 3 separate catalytic domains (CPS II, Asp-adding enzyme, ring-closing enzyme)
  • one addt’l rxn occurs on outer surface of inner mito mem

3a. attaching pyrimidine base to ribophosphate backbone

orotate + PRPP → ortidylate (OMP - precursor to UMP) [orotate phosphoribosyl transferase]

3b. converting OMP to UMP (parent pyrimidine)

OMP (ortidylate) → UMP (uridylate) [orotidylate decarboxylase]

* orotate phosphoribosyl transferase and orotidylate decarboxylase are diff members of same polypepide

  • decreased activity of either = orotic aciduria (megaloblastic anemia, lots of orotate in urine)
  • tx: supplementation with CMP, UMP, uridine to bypass metabolic block

4. synthesizing UDP and UTP

UMP → UDP → UTP via phosphorylation

  • requires specific nucleoside monophosphate kinase, nonspecific nucleoside diphosphate kinase

5. synthesizing CTP

*only way to make cytosine nt:

convert UTP + ATP + Gln→ CTP [CTP synthetase]

  • CTP inhibits CTP synthetase via feedback inhibition

***only ways to make thymine nt:

  1. from precursor, deoxyuridine monophosphate (dUMP)
  2. by deaminating deoxycytidine monophosphate (dCMP)

detour: convert ribonucleotides to deoxyribonucleotides

29
Q

orotic aciduria

A

deficiency in activity of enzymes involved in synthesizing OMP, converting OMP → UMP in pyrimidine synth

*orotate phosphoribosyl transferase and orotidylate decarboxylase are diff members of same polypepide

  • decreased activity of either = orotic aciduria

symptoms: megaloblastic anemia, lots of orotate in urine

tx: supplementation with CMP, UMP, uridine to bypass metabolic block

30
Q

converting ribonucleotides to deoxyribonucleotides

key rxn, enzyme

regulation

clinical correlation: ADA deficiency

A

need dUMP or dCMP for thymine synthesis

nucleoside diphosphates (ADP, GDP, CDP, UDP) → deoxynucleotide diphosphates (dADP, dGDP, dCDP, dUDP)

  • ribonucleotide reductase, highly expressed in proliferating cells that enter S phase
  • requires NADPH to keep catalytic site reduced

regulation

+ : binding of ATP to active site

  • : binding of dATP to active site (feedback inhibition?

which molecule binds to active site (i.e. which molecule the enzyme is reducing at a given time) is regulated by binding of other molecules to substrate specificity site

in ADA deficiency…

lack of ADA → buildup of adenosine → conversion to ribont, deoxyribont forms : high levels of dATP

  • inhibits ribonucleotide reductase → blocks production of all deoxyribonucleotides → prevents bone marrow from making B and T cells :(
31
Q

ribonucleotide reductase and ADA deficiency

A

in ADA deficiency…

  • lack of ADA → buildup of adenosine → conversion to ribont, deoxyribont forms : high levels of dATP
  • inhibits ribonucleotide reductase → blocks production of all deoxyribonucleotides → prevents bone marrow from making B and T cells :(
32
Q

synthesis of thymine nts

A

requires dUMP, formed by either…

  • deamination fo dCMP (main pathway)
  • phosphorylase action on dUTP (gets rid of excess dUTP - prevents it from getting incorp’d into DNA, where it doesnt belong)

dUMP +N5N10-methylene THF → dTMP [thymidylate synthase]

  • antitumor agent 5-fluorouracil gets converted to 5-FdUMP - covalent inhibitor of thymidylate synthase, leading to lack of thymine and cell death
  • methotrexate and others inhibit DHFR → prevent recycle THF, block purine and TMP synthesis
33
Q

pyrimidine salvage

A

purine salvage: enzymes link PRPP to base → ribonucleotides (1 step)

pyrimidine salvage requires 2 steps

1. base coupled to sugar

pyrimidine base + ribose-1P → pyrimidine nucleoside + P [nucleoside phosphorylase]

2. addition of P to nucleoside

pyrimidine nucleoside + ATP → pyrimidine nucleotide + ADP [nucleoside kinase]

  • need to know thymidine kinase : salvages nucleoside thymidine → TMP]
34
Q

acyclovir

A

guanosine analog

interferes with viral replication

  • P’d by viral thymidine kinase → preferentially incorporated into viral DNA → premature DNa chain termination
35
Q

degradation of pyrimidines

A

don’t need to know details

catabolism of pyrimidines → highly soluble products

CMP, UMP → beta-alanine

TMP → beta-aminoisobutyrate

NH3 and CO2 released in process

Gastrointestinal System (35 decks)

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