Nucelotide metabolism

why must dNTPs be made on demand

2’ hydroxyl group

ribonucleotide vs deoxyribonucleotide

NDP (two phosphates)

what level of phosphorylation does RNR act

starving tumor cells of ingredients for DNA synthesis

why would a tumor be sensitive to dTMP block

can lead to toxicity if you do not screen for a deficiency

5-FU and dihydropyrimidine dehydrogenase

reduces 2’-OH

RNR does this

phosphorylates to create dNTP using ATP

NDP-kinase does this

in animals, a thioredoxin system, but in bacteria a glutathione/glutaredoin system

bacteria vs animal reduction

spontaneous hydrolysis can happen to RNA, this makes it less stable compared to DNA

why are RNA properties different than DNA which does not have 2’-OH

sole enzyme for de novo synthesis of dNTPs; these deoxyribonucleotides are needed to repair and replicate DNA

why is RNR universally essential

R1 subunit that is catalytic and large and R2 subunit which houses radicals

RNR structure

houses stable tyrosyl (T122 in e coli or 177 in humans) free radical generated by a diferric-oxo cluster; the radical travels 35 angstroms through protein to the C site to start catalysis at cysteine on R1 subunit

R2 subunit of RNR radical mechanism

active site: ATP bound, enzyme is on, but if dATP bound, enzyme is off; specificity site which picks the substrate: when one dNTP fills, it shifts RNR toward making the others to keep all four dNTP pools roughly balanced

two switches that control RNR

deoxyribonucleotides are substrate for DNA synthesis, so we are trying to make equal amounts of both; dATP means we do not need to make any

why is RNR regulation important

competitive inhibition depends on substrate amounts and still works some; polymerization inactivates it; we want it to be highly controlled regulation

why is hexamerizaiton of RNR a more powerful off switch than substrate competition; what would happen if there was a D16R that abolishes dATP mediated inhibition?

dATP goes up, while dCTP, dGTP, and dTTP all depleted; this is because dATP will cause heximer formation and deactivate RNR

If a cell lost adenosine deaminase (ADA), dAMP, dADP, and dATP all accumulate. What would happen

RNR does not make dTDP; thymidine has a methyl group on c-5 that is not on an RNA base; we need a separate methylation step

Why does dTMP need a special pathway