pyrimidine and purine nucleotide biosynthesis Flashcards
our cells get the nucleotides they need through…
Dietary Intake of RNA & DNA – Ingested nucleic acids
are degraded through sequential action of hydrolytic
enzymes (nucleases).
Salvage of bases – Allows some cells (especially
neurons) to re-use purine bases by forming nucleotides.
de novo Synthesis – We make our own purines and
pyrimidines, especially in rapidly dividing cells
nucleotide metabolism
ATP & GTP
Coenzymes(contain adenine: CoA, NAD+, FAD, Cobalamin; made from GTP: folates)
DNA(dNTPs) & RNA (NTPs)
phosphoribosyl- pyrophosphate synthetase
Salvage and de novo synthesis pathways require PRPP to
make nucleoside-5’-monophosphate
Purine biosynthetic and salvage reactions utilize an activated
sugar intermediate: (know structure of ribose-5-phosphate)
PRPP reacts with purine and pyrimidine bases to form the corresponding nucleoside-5’-P
what do you need to know about pyrimidine biosynthesis?
dont need to know biosynthetic intermediates, but must know CHEMICAL LOGIC
how does cps-ii form carbamoyl-phosphate?
since it is a glutamine dependent reaction, it uses glutamine and ATP through a tunnel
how does carbamoyl asparate form from carbamoyl-P?
ATCase and carbamoylation is driven by Pi release
how does dihydroorotate form from carbamoyl-aspartate
-h2O (driven by electron delocalization)
how does orotate form from dihydroorotate?
hydride transfer (NAD+ to NADH)
How does orotidine 5’-monophosphate (OMP) form from orotate?
N-1 of ring attacks a-PRib-PP (with inversion of configuration)
how does OMP form UMP (uridine 5’-monophosphate)?
OMP decarboxylase rate enhancement is 1.1x10^17, putting t1/2 at 3 billion years for the uncatalyzed reaction (CO2 released)
what structure must you know concerning pyrimidine biosynthesis?
UMP or uridine 5’-monophosphate
phosphorylation of pyrimidine nucleotides
Once formed, UMP is converted to UDP and then to UTP.
All cells have “housekeeping” kinases that catalyze the
formation of pyrimidine di- and tri-phosphates
The adjective “housekeeping” emphasizes
that these enzymes help keep cells in good working order & that their rates of synthesis & degradation assure their
constant presence, at the ready to meet the cell’s needs
CTP (a structure you must know) is formed directly from…
UTP
two different CTP synthase isoforms:
Human CS-I – essential for CDP-diacylglycerol formation
Human CS-II – rate-limiting enzyme in biosynthesis of
pyrimidine precursors of RNA and DNA
§ Shows cooperativity in v versus [UTP] plots
§ Activated by GTP (a purine), thereby acts to balance
amounts of purine and pyrimidine nucleotides.
§ CS-II deficiency affects cell growth and development
what potently inhibits thymidylate synthase?
fluorouracil
____________&______________ are folate-like inhibitors of these rxns (targets for cancer chemotherapy)
methotrexate and aminopterin
5- fluorouracil: cancer chemotherapy
bioactivation of prodrug (5FU) forms the active drug (5-FdUMP); FdUMP forms covalent adduct with thymidylate synthase, blocking methyl transfer from methylene-THF to 5-position of dUMP. without TMP theres no DNA for cells to multiply
methotrexate: treatment of cancer & rheumatoid arthritis
MTX inhibits dihyrofolate reductase (DHFR), an enzyme required TMP biosynthesis. no TMP-> no DNA-> no growth
pathway for purine biosynthesis
Liver & immune cells primarily biosynthesis purines.
Starts with PRPP, building purine ring on the ribose ring
Requires great input of energy
One-carbon transfers are provided
by N10 -formyl-tetrahydrofolate.
Inosine 5’-P (IMP) is first fully formed purine nucleotide
how do you form phosphoribosylamine from PRib-PP?
glutamine hydrolysis; NH3 transfer; followed by displacement of PPi (Gln-> <-Glu+PPi)
formation of glycinamide ribonucleotide (GAR) from PRib-NH2
glycyl-P intermediate forms, followed by amide bond formation (Gly +ATP-> <- ADP+Pi)
formation of formylglycinamide ribonucleotide (FGAR) from GAR
formyl transfer to amino group (formyl-THF-> <- THF)
formation of formylglycinamidine ribonucleotide from FGAR
- glutamine hydrolysis
- :NH3 Transfer
- :NH3 displaces Pi
(Gln+ATP-> <-ADP+Pi)
Formation of aminoimidazole
ATP<->ADP+Pi; ring closure, phosphorylation activates loss of oxygen
formation of carboxyaminoimidazole ribonucleotide
HCO3- + ATP<-> ADP+Pi; carboxylation occurs via carbanion attack on carboxyl-P intermediate
formation of succinyl-CAIR
Asp+ATP<-> ADP+Pi; ATP first forms acyl-P; then amino of aspartate attacks to displace Pi
formation of AICAR
lyase-type reaction<-> catalyzes trans elimination
formation of formyl-aminoimidazole-carboxamide ribonucleotide
formyl-THF<->THF (formyl transfer to amino group)
formation of inosine 5’-monophosphate
-H2O<->+H2O (driven by resonance ie aromatic system is highly stabilized by electron delocalization)
what provides energy for AMP synthesis?
GTP
what do you need to know the steps of conversion for?
IMP to AMP
adenylosuccinate synthetase mechanism
- covalent intermediate (introduces good leaving group) 2. Aspartate displaces Pi
key points: IMP tautomerizes to form -OH at C6 GTP phosphorylates IMP to form the 6-phospho-IMP intermediate. Aspartate amino group displaces phosphate
what do you need to know the steps of conversion from IMP to …
GMP
mechanism for GMP synthase
- adenylylation of the purine -OH group 2. glutamine hydrolysis & :NH3 transfer through tunnel 3. nucleophilic attack :NH3 to form GMP
control of purine nucleotide synthesis
- AMP inhibits AMP-Succ synthetase, thereby directing IMP to GMP
- GMP inhibits IMP dehydrogenase, thus directing IMP to AMP
- high IMP feedback inhibits 5’-P-Rib-NH2 formation
- high AMP & GMP feedback inhibit PRPP synthetase
what needs to happen when adenine nucleotides are in excess?
rebalancing purine nucleotide synthesis (look at slide 16)
what needs to happen when guanine nucleotides are in excess?
rebalancing purine nucleotide synthesis (look at slide 17)
purine salvage pathway
hypoxanthine guanine phosphoribosyl transferase; HGPRT salvages purines from RNA & DNA degradation; Adenine is NOT salvaged by HGPRT-> adenine is most abundant purine & high levels of adenine would block IMP and GMP re-synthesis, adenine deaminase converts adenine to hypoxanthine, IMP formation allows cells to rebalance AMP & GMP
what do you need to know the pathway for on slide 19 concerning purines?
purine degradation pathway
gout
pathologic precipitation of sodium urate; at neutral pH, sodium urate accumulates to supersaturating concentrations & then crystallizes; crystals accumulate in joints, causing severe inflammation and pain; crystals activate inflammation, stimulating invasion by white blood cells, which attack foreign particles, stimulating formation of even more crystals; gout is treated with allopurinol (xanthine oxidase inhibitor) & excess hypoxanthine & xanthine are harmlessly excreted in urine
ribonucleotide reductase (RNR)
make 2’-deoxy-ribonucleotides needed for DNA synthesis-> indirectly transfers electrons from NADPH to NDP substrate; in eukaryotes, RNR substrates are ADP,GDP,CDP,&UDP; in prokaryotes, RNR substrates are ATP,GTP,CTP,&UTP; ‘ribo-TDP’ & ‘ribo-TTP’ are not RNR substrates-> there are only deoxyribo-forms of TMP, TDP, &TTP, thymidylate synthase was last step in transition to DNA world
RNR regulation
RNR must achieve the right mix of dNTPs needed for DNA synthesis by DNA polymerase; DNA polymerase requires roughly the same concentration of dNTPs, despite disparate cellular concentrations of ribo-NDPs; dNDP formation is regulated by allosteric interactions; RNR achieves balanced synthesis of dADP,dGDP,dUDP,& dCDP
what are structures you must know?
a-ketoglutarate, oxaloacetate, pyruvate, y-glutamyl-phosphate, carbomoyl-phosphate, uric acid, urea, ornithine, citrulline, arginino-succinate, NAD+ & NADH, phenylpyruvate, s-adenosylmethionine, tri-iodthyronine, PRPP, adenosine mono/di/tri-P, guanosine mono/di/tri-P, inosine monophosphate (IMP), cytosine mono/di/tri-P, uridine mono/di/tri-P, thymidine mono/di/tri-P
what are other structures you must know?
adenine, guanine, cytosine, uracil, thymine
