pyrimidine and purine nucleotide biosynthesis

Question 1

our cells get the nucleotides they need through…

Answer 1

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

Question 2

nucleotide metabolism

Answer 2

ATP & GTP
Coenzymes(contain adenine: CoA, NAD+, FAD, Cobalamin; made from GTP: folates)
DNA(dNTPs) & RNA (NTPs)

Question 3

phosphoribosyl- pyrophosphate synthetase

Answer 3

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

Question 4

what do you need to know about pyrimidine biosynthesis?

Answer 4

dont need to know biosynthetic intermediates, but must know CHEMICAL LOGIC

Question 5

how does cps-ii form carbamoyl-phosphate?

Answer 5

since it is a glutamine dependent reaction, it uses glutamine and ATP through a tunnel

Question 6

how does carbamoyl asparate form from carbamoyl-P?

Answer 6

ATCase and carbamoylation is driven by Pi release

Question 7

how does dihydroorotate form from carbamoyl-aspartate

Answer 7

-h2O (driven by electron delocalization)

Question 8

how does orotate form from dihydroorotate?

Answer 8

hydride transfer (NAD+ to NADH)

Question 9

How does orotidine 5’-monophosphate (OMP) form from orotate?

Answer 9

N-1 of ring attacks a-PRib-PP (with inversion of configuration)

Question 10

how does OMP form UMP (uridine 5’-monophosphate)?

Answer 10

OMP decarboxylase rate enhancement is 1.1x10^17, putting t1/2 at 3 billion years for the uncatalyzed reaction (CO2 released)

Question 11

what structure must you know concerning pyrimidine biosynthesis?

Answer 11

UMP or uridine 5’-monophosphate

Question 12

phosphorylation of pyrimidine nucleotides

Answer 12

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

Question 13

CTP (a structure you must know) is formed directly from…

Answer 13

UTP

Question 14

two different CTP synthase isoforms:

Answer 14

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

Question 15

what potently inhibits thymidylate synthase?

Answer 15

fluorouracil

Question 16

____________&______________ are folate-like inhibitors of these rxns (targets for cancer chemotherapy)

Answer 16

methotrexate and aminopterin

Question 17

5- fluorouracil: cancer chemotherapy

Answer 17

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

Question 18

methotrexate: treatment of cancer & rheumatoid arthritis

Answer 18

MTX inhibits dihyrofolate reductase (DHFR), an enzyme required TMP biosynthesis. no TMP-> no DNA-> no growth

Question 19

pathway for purine biosynthesis

Answer 19

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

Question 20

how do you form phosphoribosylamine from PRib-PP?

Answer 20

glutamine hydrolysis; NH3 transfer; followed by displacement of PPi (Gln-> <-Glu+PPi)

Question 21

formation of glycinamide ribonucleotide (GAR) from PRib-NH2

Answer 21

glycyl-P intermediate forms, followed by amide bond formation (Gly +ATP-> <- ADP+Pi)

Question 22

formation of formylglycinamide ribonucleotide (FGAR) from GAR

Answer 22

formyl transfer to amino group (formyl-THF-> <- THF)

Question 23

formation of formylglycinamidine ribonucleotide from FGAR

Answer 23

1. glutamine hydrolysis
2. :NH3 Transfer
3. :NH3 displaces Pi
   (Gln+ATP-> <-ADP+Pi)

Question 24

Formation of aminoimidazole

Answer 24

ATP<->ADP+Pi; ring closure, phosphorylation activates loss of oxygen

Question 25

formation of carboxyaminoimidazole ribonucleotide

Answer 25

HCO3- + ATP<-> ADP+Pi; carboxylation occurs via carbanion attack on carboxyl-P intermediate

Question 26

formation of succinyl-CAIR

Answer 26

Asp+ATP<-> ADP+Pi; ATP first forms acyl-P; then amino of aspartate attacks to displace Pi

Question 27

formation of AICAR

Answer 27

lyase-type reaction<-> catalyzes trans elimination

Question 28

formation of formyl-aminoimidazole-carboxamide ribonucleotide

Answer 28

formyl-THF<->THF (formyl transfer to amino group)

Question 29

formation of inosine 5'-monophosphate

Answer 29

-H2O<->+H2O (driven by resonance ie aromatic system is highly stabilized by electron delocalization)

Question 30

what provides energy for AMP synthesis?

Answer 30

GTP

Question 31

what do you need to know the steps of conversion for?

Answer 31

IMP to AMP

Question 32

adenylosuccinate synthetase mechanism

Answer 32

1. 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

Question 33

what do you need to know the steps of conversion from IMP to ...

Answer 33

GMP

Question 34

mechanism for GMP synthase

Answer 34

1. adenylylation of the purine -OH group 2. glutamine hydrolysis & :NH3 transfer through tunnel 3. nucleophilic attack :NH3 to form GMP

Question 35

control of purine nucleotide synthesis

Answer 35

1. AMP inhibits AMP-Succ synthetase, thereby directing IMP to GMP 2. GMP inhibits IMP dehydrogenase, thus directing IMP to AMP 3. high IMP feedback inhibits 5'-P-Rib-NH2 formation 4. high AMP & GMP feedback inhibit PRPP synthetase

Question 36

what needs to happen when adenine nucleotides are in excess?

Answer 36

rebalancing purine nucleotide synthesis (look at slide 16)

Question 37

what needs to happen when guanine nucleotides are in excess?

Answer 37

rebalancing purine nucleotide synthesis (look at slide 17)

Question 38

purine salvage pathway

Answer 38

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

Question 39

what do you need to know the pathway for on slide 19 concerning purines?

Answer 39

purine degradation pathway

Question 40

gout

Answer 40

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

Question 41

ribonucleotide reductase (RNR)

Answer 41

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

Question 42

RNR regulation

Answer 42

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

Question 43

what are structures you must know?

Answer 43

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

Question 44

what are other structures you must know?

Answer 44

adenine, guanine, cytosine, uracil, thymine