Nucleotide Metabolism - Roth 3/14/16 Flashcards
roles of nucleotides in the body
- building blocks of nucleic acids (DNA, RNA)
- energy currency in the cell (ATP, GTP)
- carriers of activated intermeds (UDP-glucose, SAM)
- structural components of essential cofactors (NAD+)
- metabolic regulation, signal molecules (cyclic AMP)
nucleotide nomenclature
base (AGCU, T)
nucleoside: base + sugar (adenosine, guanosine, cytidine, uridine, thymidine)
nucleotide: base + sugar + P (_TP, _DP, _MP)
sources of nucleotides
- de novo synthesis
* base built from scratch - salvage pathways
- reuse “preformed bases” to build nts (obtained from nucleic acid breakdown in cells or dietary nucleic acids)
- used often in
nucleotide degradation overview
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)
de novo synthesis of purine nts
- synthesis of sugar-P backbone
sugar-P backbone (aka PRPP)
ribose-5P + ATP → PRPP
-
PRPP synthetase
- + : inorganic P
- : purine ribonucleotides (ADP, ATP, GDP, GTP)
de novo synthesis of purine nts
- committing PRPP to purine synthesis
committed step for making purines
PRPP + Gln → PRA (5-phosphoribosylamine)
-
PRPP-amino-transferase
- + : PRPP
- : AMP, GMP, IMP (parent molecule for AMP/GMP)
glutamine analogs
clinically, antibiotics azaserine and DON are Gln analogs
- irreversibly inhibit PRPP-amin-transferase → prevent committed step of purine synth!
synthesis of IMP
IMP is the parent structure for AMP, GMP
PRPP + Gln → IMP
- need energy (ATP)
- carbon sources (THF, CO2)
- amino acid (Gln, Asp, Gly)
importance of THF to purine synthesis
role of methotrexate and aminopterin
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)
importance of THF to purine synthesis
role of sulfonamides
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
purine synthesis
IMP → AMP
IMP + GTP (energy) + Asp (N source) → adenylosuccinate
adenylosuccinate → fumarate + AMP
*GTP is needed to synthesis AMP
*AMP demonstrates feedback inhibition
purine synthesis
IMP → GMP
IMP + NAD+ → XMP
- IMP DH (inhibited by ribavirin, high GMP)
XMP + ATP (egy) + Gln (N source) → GMP
inhibitors of IMP dehydrogenase
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
regulation of purine biosynthesis:
what determines whether IMP → GMP or AMP?
reciprocity
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
adding phosphates
conversion of NMP → NDP → NTP
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
point of reciprocity in purine synthesis
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
purine salvage pathway
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
two salvage pathways (and key enzymes)
1. APRT (adenine phosphoribosyl transferase)
- adenine + PRPP → AMP
2. HGPRT (hypoxanthine-guanine phosphoribosyl transferase)
- guanine + PRPP → GMP
- hypoxanthine + PRPP → IMP
what happens when salvage pathways are blocked?
de novo synthesis is turned on!
water/tap/drain? look at slide
Lesch-Nyhan syndrome
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
degradation of purines to uric acid
- 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] - removal of amino group from adenosine → inosine [adenosine deaminase]
- removal of ribose from inosine and/or guanosine [purine nucleoside phosphorylase, PNP]
- inosine → hypoxanthine
- guanosine → guanine
- hypoxanthine, guanine → xanthine → uric acid
- 2x xanthine oxidase
- uric acid can be an antioxidant, excreted in urine
adenosine deaminase deficiency
→
severe combined immunodeficiency (SCID)
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
adenosine deaminase deficiency
leads to accumulation of DATP (50x higher conc than normal)
potential explanations for effects on B and T cells…
- high [DATP] shuts down ribonucleotide reductase, stops dNTP synth/DNA synth
- high [deoxyadenosine] shuts down S-adenosylhomocysteine hydrolase - req for methylation of RNA/DNA bases
- high [adenosine] → high cAMP levels
*no suitable explanation for effects ltd only to B and T cells :(
PNP deficiency
genetic deficiency of PNP
commonly presents in childhood
symptoms: recurrent bacterial/viral/opportunistic infections
pathophys: severe deficit of T lymphocytes
tx: bone marrow transplant
uric acid
gout
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
xanthine and hypoxanthine are ore soluble than uric acid and can be excreted
why bother making uric acid in the first place?
urate in serum is also an antioxidant → scavenges ROS
purine nucleotide cycle
- anaplerotic rxn
- where is this rxn taking place
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
de novo pyrimidine synthesis
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:
- from precursor, deoxyuridine monophosphate (dUMP)
- by deaminating deoxycytidine monophosphate (dCMP)
detour: convert ribonucleotides to deoxyribonucleotides
orotic aciduria
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
converting ribonucleotides to deoxyribonucleotides
key rxn, enzyme
regulation
clinical correlation: ADA deficiency
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 :(
ribonucleotide reductase and ADA deficiency
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 :(
synthesis of thymine nts
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
pyrimidine salvage
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]
acyclovir
guanosine analog
interferes with viral replication
- P’d by viral thymidine kinase → preferentially incorporated into viral DNA → premature DNa chain termination
degradation of pyrimidines
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
