Nucleic Acids Flashcards
what is the difference between the de novo pathway and the salvage pathway?
- de novo: synthesis of nucleotides from starting material (AAs, ribose, CO2, …) and highly conserved process in eukaryotes
- salvage: recovery of bases for making new nucleotides and more divergent/varies process
what are the two de novo pathways?
- purine pathway
- pyrimidine pathway
what molecules/substrates are needed for the purine pathway to make IMP?
PRPP, glutamine, aspartate, CO2, glycine, formate
what molecules/substrates are needed for the pyrimidine pathway to make UMP?
CO2, glutamine, aspartate, PRPP
what is a base?
the aromatic part of a nucleotide
what does a molecules ending with “-side” contain?
a base + sugar (like ribose)
what does a molecules ending with “-tide” contain?
base + phosphosugar
what is the difference between a purine and pyrimidine?
- purine: two rings
- pyrimidine: 1 ring
what are the two types of sugar and what differentiates the two?
- ribose (for RNA): has an extra 2’OH
- deoxyribose (for DNA): doesn’t have 2’OH
nucleotide sugars are __ sugars in ____ conformation
D ; furanose
bases are linked to sugars by which type of bond and is that above or below?
B glycosidic bond, which is above
where is the sugar linked on a purine?
9-N
where is the sugar linked on a pyrimidine?
1-N
how are nucleotides numbered? How does it differ for purines and pyrimidines?
- primed numbers are given to the sugar ring (starting where it is linked to base)
- unprimed numbers given to bases
- pyrimidines: nitrogen at 1, 3 (not 5)
- purine: nitrogen at 1, 3, 7, 9 (not 5)
caffeine and theobromine are derivatives of which nucleotide?
xanthine
what is the de novo synthesis steps of purines?
the purine ring is assembled on the ribose phosphate
- 5-phosphoribose to PRPP
- PRPP to 5-phosphoribosylamine
- then 11 steps to make IMP
how many high energy phosphate bonds are needed for de novo purine synthesis?
7
is the pyrophosphate at the alpha or beta position on the ribose?
alpha
what is the committing step of de novo purine synthesis? what happens during this step?
- step 2 (conversion of PRPP to 5-phosphoribosylamine)
- anomeric conversion (forms b anomer)
- PPi released (so irreversible)
what donates the amine group in the second step of purine de novo synthesis?
glutamine
what are the different nitrogen donor and which part specifically is used?
- side chain of glutamine
- backbone of aspartate
how is the first ring of the purine synthesized (de novo)?
- start from 5-phosphoribosylamine
- addition of glycine
- N added from glutamine
- C from THF
- cyclization
how is the 2nd ring of the purine synthesized? what is the product?
- addition of HCO3-
- N from aspartate added in two steps ( N-succinyl adduct and release of fumarate)
- C from THF
- ring closed by dehydration (no ATP required)
- product is IMP
inosine monophosphate (IMP) is the branch point for ___________
AMP and GMP synthesis
how is AMP synthesized from IMP?
uses aspartate and GTP to add amine
how is GMP synthesized from IMP?
via XMP uses glutamine and ATP to add amine
what is the general rule of nucleotide synthesis?
amino groups put on/take off via carbonyls
how are diphosphates made?
via base specific kinases:
- adenylate kinase: AMP+ATP <=> 2ADP
- uridylate kinase: UMP+UTP <=> 2UDP
how are triphosphates made?
via non-specific kinases
- nucleoside diphosphate kinase: NDP + N’TP <=> NTP + N’DP
what is the G of the formation of di- and tri-phosphates? what does this mean?
~0, meaning the reactions are driven by concentration gradient
how do the amount of the following (intracellular concentrations) compare: RNA, NTP, dNTP, NDP, DNA, NMP (not necessarily in this order)
- 7x more RNA than DNA
- 7x more NTP than dNTP
- 7x more NTP than NDP
- 7x more NDP than NMP
how is the de novo purine synthesis regulated?
- feedback inhibition by nucleotides
- feedforward by PRPP
- inhibition and stimulation for AMP and GMP synthesis
- regulation at steps 1 and 2
how many times in the de novo purine pathway is folate used?
2
where does THF come from?
from folic acid
what are the possible different oxidation states and chemical forms of carbon units carried by THF?
oxidation:
1. methanol
2. formaldehyde
3. formate
chemical:
- methyl
- methylene
- formyl
- formimino
- methenyl
what are the following carbon units carried by THF used in: N6-methyl-THF, N5,N10-methylene-THF, N10-formyl-THF
- N6-methyl-THF: methionine cycle
- N5,N10-methylene-THF: thymidylate synthesis
- N10-formyl-THF: purine de novo
why is DNA the genetic material?
- removing the 2’OH makes the DNA phosphodiester backbone more stable
- Thymine allows spontaneous deamination of cytosine to be detected
is DNA or RNA more reduced?
DNA (1 less OH)
the synthesis of deoxyribose is carried out at which level?
DNPs (diphosphates)
how are NDP reduced to dNDP?
- reducing equivalent originate from NADPH
- goes through a series of thiols, where cysteine residues are oxidized/reduced:
1. thioredoxin reductase
2. thioredoxin
3. ribonucleotide reductase (RR) - then NDP reduced to dNDP
what are the two types of subunits of ribonucleotide reductase?
- regulator
- catalytic
what are the two allosteric sites on ribonucleotide reductase? what do they control?
- controls activity: binds ATP (activates) /dATP (inhibits)
- controls specificity: dATP/dGTP/dTTP
why is ribonucleotide reductase an unusually stable enzyme? what can destabilize it?
- due to tyrosine free radicals
- inhibited by hydroxyurea
what makes ribonucleotide reductase a good therapeutic target?
- lots of variation with species
- so, could specifically inhibit DNA synthesis
how is riboreductase regulated?
two types of feedback regulation:
- negative feedback (either specific to that nucleotide or can influence other types)
- positive: dNTPs can affect production of other types of dNTPs
incorporation of uracil into DNA induces _________
DNA repair enzymes
how does the body try to prevent incorporation of dUTP in DNA?
presence of UTPase hydrolyzes dUTP to dUMP (which is then converted to dTMP by thymidylate synthase) to decrease its concentration and prevent its incorporation
how does purine and pyrimidine de novo synthesis differ?
- purine: PRPP made first then ring assembled on it
- pyrimidine: ring assembled separately then PRPP added
explain the de novo UMP synthesis?
- channeling of steps 1,2,3:
1. make carbamoyl phosphate with CPSII
2. make carbamoyl aspartate with ATCase (aspartate added to carbamoyl phosphate)
3. makes dihydroorotase
- is at mitochondrial membrane: makes orotate
- channeling of steps 5,6
5. make OMP
6. make UMP (remove COO- group)
loss of steps 5 or 6 in pyrimidine synthesis leads to ______
orotic aciduria
how is pyrimidine de novo synthesis regulated?
- step 1 (CPSII) for animals: compartmentalization
- step 2 (aspartate transcarboxylase) for bacteria: allosteric regulation – activated by ATP and inhibited by CTP
At what level is CTP made?
Tri phosphate
How is CTP made?
- from UTP
- N from glutamine
- energy from ATP
How is the pyrimidine de novo pathway regulated?
- inhibition by pyrimidine nucleoside triphosphate
- activation by ATP and PRPP
- both either first or second step depending of organism
thymidylate synthesis is done at which level?
monophosphate
what does thymidylate synthase do?
synthesis of dTMP from dUMP
what is the methyl donor for dTMP synthesis by thymidylate synthase? what is the end-product?
Methylene-THF, released as DHF
what is the role of tetrahydrofolate? why is it important?
- to reduce DHF to THF
- cell would run out of THF due to dTMP synthesis
what is the thymidylate synthase cycle?
- THF is oxidized to DHF (produces dTMP from dUMP)
- DHF reduced by DHF reductase
- methylene group added from serine
what is FdUMP?
a suicide inhibitor that can be used to target infections or cancer (it covalently modifies thymidylate synthase so it stops synthesis)
what are different types of inhibitors of DHF reductase and what do they all do?
- all inhibit DNA synthesis
- THF analogs, competitive inhibitor, anti-cancer, anto-bacterial
what is the principle of the salvage pathway?
recovery of bases for making new nucleotides
what is Lesch-Nyhan disease?
- genetic loss of HGPRT activity (cannot salvage hypoxanthine and guanine)
- symptoms: gout and mental retardation
what is HGPRT and what is its role?
- hypoxanthine/guanine phosphoribosyl transferase
- converts either: hypoxanthine + PRPP to inosinate or guanine + PRPP to guanylate
how can nucleosides be formed in the salvage pathway? are these specific?
- nucleotide phosphorylase
- nucleotide transglucosylase (exchange bases)
- they are specific to avoid making thymine ribonucl.
what are the salvage pathway enzymes?
- phosphoribosyl transferase (e.g. making OMP with orotic acid)
- nucleotide phosphorylase
- nucleoside kinase
- nucleotide transglucosylase (exchange bases)
- deaminases
- nucleoside monophosphate kinase
- nucleoside diphosphate kinase
how are purines degraded?
funnel downwards:
- nucleotides (AMP, IMP, XMP, GMP)
- nucleosides (adenosine, inosine, xanthosine, guanosine)
- bases, xanthine
- uric acid
what results in loss of adenosine deaminase?
SCID
what is a treatment for gout?
inhibit xanthine oxidase
what is gout? what causes it?
- deposition of uric acid in joints
- symptomatic of metabolic imbalance (excess de novo purine synthesis like high levels of PRPP)
how do birds eliminate nitrogen? why?
by making uric acid in order to conserve water since what we do (ammonia) takes a lot of water
how are pyrimidines degraded?
- nucleotides (CMP, UMP/dTMP)
- nucleosides (cytidine, uridine/deoxythymidine)
- uracil or thymine
- deaminated to carbonyls and ring reduced
