Lecture 12 (part 2): Pyrimidine Synthesis

in pyrimidine synthesis; the ring is build _______ then _______ to ribose

first; attached

what molecule make up the pyrimidine ring?

Gln; HCO3-; Asp

what molecule do carbons 5, 6 and nitrogen 1 come from?

aspartate

what molecule does nitrogen 3 come from?

glutamine amide

what molecule does carbon 2 come from?

HCO3-

what enzyme catalyzes the first step of pyrimidine synthesis? (to form the ring)

CPS II

CPS-II combines _______ and ________ to form __________

HCO3; Gln amide; carbamoyl-phosphate

in the second step of pyrimidine synthesis; carbamoyl phosphate reacts with _________ to form _________

aspartate; carbamoyl-aspartate

what enzyme forms carbamoyl aspartate?

aspartate transcarbamylase

what happens to carbamoyl aspartate in pyrimidine synthesis?

ring closes

what molecule forms from carbamoyl aspartate ring closing?

dihydroorotate

what does dihydroorotate form?

orotate

what is the difference in structure between orotate and dihydroorotate?

double bond in the ring

once orotate is formed, it reacts with _________ to form ________

PRPP; OMP

what does OMP stand for?

orotine monophosphate

what molecule forms when OMP undergoes a decarboxylation?

UMP

what is the reaction pathway for pyrimidines? (not a specific base)

HCO3 + Gln = carbamoyl-phosphate + aspartate = carbamoyl aspartate → dihydroorotate → orotate → OMP → UMP

UMP is first converted into _______ and then ______

UTP; CTP

what enzyme converts UMP to UDP?

UMP kinase

is NDK base specific?

no, works on all bases

is UMP kinase base specific?

yes, each base has their own kinase

what does NMP kinase do?

add a second phosphate

what does NDK do?

add a third phosphate

what enzyme converts UTP to CTP?

CTP synthase

UTP is converted into CTP using a molecule of _______. The enzyme ________ replaces the ______ group with a ________ group

glutamine; CTP syntheses ketone; amino

does synthesizing CTP from UTP require an ATP?

yes, 1 ATP

what enzymes are CAD made up of? (3)

CPSII; ATCase; DHOase

what is DHOase?

dihydrooritase (oxidizes)

what 2 functions does UMP synthase do?

make OMP, decarboxylate to UMP

what 2 molecules activate CAD activity?

PRPP and ATP

ATP and PRPP activating CAD is an example of

substrate activation

what 3 molecule inhibit CAD?

UDP, UTP, CTP

UDP, UTP and CTP inhibiting CAD is an example of _________

product inhibition

CTP synthase is activated by ________ and inhibited by _______

GTP; CTP

what is the first step of nuceloside Triphosphate synthesis?

base specific kinases generate NDPs

what is the second step of nucleoside Triphospahte synthesis?

NDK generate NTPs

base specific kinases generate _______

NDPs

NDKs generate _______

NTPs

what enzyme converts GMP to GDP?

GMP kinase

what enzyme converts AMP to ADP?

adenylate kinase

what enzyme converts UMP to UDP?

UMP/CMP kinase

what drives the reaction of NMPs to NDPs?

ATP hydrolysis (and high [ATP])

most of the carbon flux in nucleotide metabolism generates _______(NTPs or dNTPs)

NTPs

most cells have much more __________ than _________

RNA; DNA

what are the only 2 purposes of dNTPs?

DNA synthesis and repair

the main difference between dNTPs and NTPs are a missing __________ at the ________ carbon

hydroxyl; 2’

what base is ONLY found as a dNTP?

Thymine

does the cell synthesize dUTP?

yes, but rapidly converted to dTTP

what enzyme synthesize dNTPs?

RNR

what does the enzyme RNR do?

remove hydroxyl group from NTPs (synthesizes dNTPs)

how many phosphates do the nucleotides have when they are used for making deoxygenated nucleotides by RNR?

2 (NDPs)

After RNR removes a hydroxyl group, the dNDPs undergo addition of a ____________ to form _________ by the enzyme ________

phosphate; dNTPs; NDK

what type of mechanism is used by RNR?

free radial mechanism

what could happen if a proper balance of dNTPs are maintained in the cell

increase mutations; lethal

how many subunits does RNR have?

4

RNR stand for?

ribonuclotide reductase

RNR has 2 ______ subunits and 2 ______ subunits

alpha; beta

the beta subunit of RNR is the ___________ protein

radical generating

the alpha subunit of RNR is the ________ protein

nucleotide-binding

EACH alpha-subunit has ______ catalytic site; and _______ allosteric site

1; 2

the beta subunit of RNR generates a _________ at the B site

tyrosine radical

the free radical generated by the B subunit of RNR is then used at the ______________ site

alpha-active

RNR balances the synthesis of dNTPs via __________ binding of _______ at the _________ site

sequential; dNTPs; specificity

what binds at the specificity site of RNR?

dNTPs

what binds to the active site of RNR?

ATP and dATP

when ATP is bound to RNRs active site, the enzyme is _________

ON

when dATP is bound to RNRs active site, the enzyme is _________

OFF

RNR uses _______ catalysis

sequential

when first turning on RNR, _______ binds to the active site, and ______ binds to the specificity site

ATP; ATP also

what binds to the catalytic site?

NDP substrate

once RNR forms the product, they _______ the enzyme and are converted into the _______ form, which will regulate RNR at the ________ site

leave; dNTP; specificity

what molecules regulate RNR (general)

dNTPs

how do the products of RNR regulate the enzyme’s activity?

they bind to specificity site

increasing concentrations of dATP will ________ ATP in the activity site and turn the enzyme ________

replace; OFF

what is the order of synthesis dNDP synthesis for RNR?

dUDP/dCDP, dGDP, dATP

starting from UMP, we need _____ enzymes to synthesize dTTP

5

what enzyme converts UMP to UDP?

ribonucleotide kinase (UMP kinase)

what converts UDP to dUDP?

RNR

what converts dUDP to dUTP?

NDK

what is dUTP immediately converted to?

dUMP

why is dUTP immediately converted into dUMP instead of dTTP?

very reactive, want to get rid of it quickly to prevent buildup

what enzyme converts dUTP to dUMP?

dUTP phosphatase (dUTPase)

if the concentration of dUTP is too high, it could be:

incorporated into DNA

what enzyme is used to convert dUMP to dTMP?

thymidylate synthase

what enzyme converts dTMP to dTDP?

thymidylate kinase

what enzyme converts dTDP into dTTP?

NDK

why does dUTP → dUMP occur so quickly?

hydrolysis of PP (very energetically favorable)

how does thymidylate synthase convert dUMP to dTMP?

adds a methyl group

what cofactor is used by thymidylate synthase?

THF

where does the methyl group in dTMP come from?

N5,N10-methylene THF

the synthesis of dTMP is connected to _________ metabolism and ________

amino acid; one carbon metabolism

what enzyme is used to regenerate THF after donating a methyl group to dTMP?

dihydrofolate reductase

nucleoside bases are degraded to _______ and _______

uric acid; urea

nucleic acids are decomposed into oligonucleotides by ________

endonuclease

oligonucleotides are decomposed into NMPs by _________

exonuclease

NMPs are converted into nucleosides (remove phosphate) by _________

nucleotidases

what enzyme removes the ribose group from nucleosides?

nucleoside phosphorylase

purine bases are degraded into _________

uric acid

pyrimidine bases are degraded into _______

urea

endonuclease convert _________ into _________

nucleic acids; oligonucleotides