what is the first pyrimidine made
UMP -> UDP -> UTP -> CTP via carbamyl phosphate
what is CPS-II
1. where is it located
2. what inhibits it
3. what activates it
4. what is its source of N
cabamoyl phosphate synthetase II: enzyme that catalyzes the 1st step of pyrimidine synthesis
1. found in the cytosol of all tissues
2. inhibited by UMP or UTP = end product inhibition
3. activated by PRPP
4. glutamine supplies its Nitrogen
first step of pyrimidine synthesis?
CPS-II catalyzes reaction
glutamine + CO2 + ATP -> carbamoyl phosphate + glutamate
UTP or UMP end-product inhibits this rxn; PRPP activates it
what inhibits or activates the 1st enzyme of pyrimidine synthesis?
PRPP activates it
UTP inhibits it
what is 2nd step of pyrimidine synthesis?
rate limiting step: aspartate transcarbamoylase does reaction:
carbamoyl phosphate + aspartate -> N-carbamoylaspartate
inhibited by production of CTP, stimulated by ATP
what is rate limiting enzyme of pyrimidine synthesis?
what inhibits or stimulates it?
2nd step of the synthesis pathway
end-product inhibited by CTP, stimulated by ATP
what is orotate?
an intermediate in the pyrimidine synthesis pathway
the most abundant deficiency in urea cycle = ornithine transcarbamylase, causes high levels of orotate in the blood
what might cause high levels of orotate in the blood? why?
deficiency in ornithine transcarbamylase of the urea cycle
if the carbamoyl phosphate made in the 1st step of the urea cycle isn't used b/c of a deficiency in ornithine transcarbamylase that leaves it not synthesized into orotate, it'll make orotate leave the cyto, cross into the mito, and get excess of orotate in the blood
how are nucleoside mono/di/tri phosphates interconvertable?
via the nucleoside monophosphate kinases or nucleoside diphosphates that're specific for each base
nucleoside monophosphate kinases:
GMP + ATP GDP + ADP or CMP + AMP CDP + ADP
GDP + ATP GTP + ADP or CDP + ATP CTP + ADP
how can a deoxynucleotide be converted to a ribonucleotide?
it's not possible to convert a deoxynucleotide -> ribonucleotide but a ribonucleotide can -> deoxynucleotide by ribonucleotide reductase
function of ribunucleotide reductase?
catalyzes converstion of ribose to deoxyribose base for DNA
structure of ribunucleotide reductase?
2 major subunits, B1 and B2
activity site &
substrate specificity site &
substrate binding site
function of the overall activity site?
what can bind to it, what's the effect?
overall activity site:
where ATP binds and stimulates the enzyme
thus controls activity of the whole enzyme
if dATP binds, it shuts off the enzyme
what controls activity of the ribunucleotide reductase enzyme? how?
ATP binds activity site -> sitmulates synthesis of deoxyribonucleotides
dATP binds activity site -> prevents more synthesis of deoxynucleotides
what function of substrate specificity site?
what binds here determines which ribonucleotide diphosphate will be converted to the deoxyribonucleotide diphosphate at the enzyme's activity site
function of substrate binding site?
where substrate binds
substrate is ribonucleotide diphosphate (XDP) which through the RR enzyme becoase doxyribunucleotide diphosphate (dXDP)
what's the interplay between purines and pyrimidines in the RR activation?
if ATP, a purine, binds to the substrate specificity site, it activates RR so that it converts pyrimidine substrates to deoxypyrimidines
structure of B2 subunit of ribonucleotide reductase?
has iron oxygen catalytic centers
has a Y* radical that's formed in the catalytic mechanism
how does the ribonucleotide reductase system work?
ATP binds to the overall activity site, turns on the enzyme
substrate binds to the B2 subunit, activating the Fe-O centers
B2 generates a Tyrosine radical
OH that was on C2 gets removed as H2O, generating a radical carbonium ion
Now add an H from the SH groups of B2 to the Carbonium ion
Regenerate teh H of the SH group
is H from the C3 ever lost in the RR reaction?
what's the function of thioredoxin and thioredoxin reductase?
enzymes with SH groups that regenerate the SHs of the ribonucleotide reductase used in catalytic mechanisms
it thus becomes oxidized to oxidized thioredoxin
thioredoxin reductase then reduces oxidized thioredoxin to restore SH to it
FADH2 finally reduces the oxidized thioredoxin reductase sulfurs to their SH form and makes FAD
NADPH reduces FAD to make NADP+ and regenerate FADH2
how could chemotherapy work with the RR system of enzymes?
1) inhibit the free radical produced in the RR B2 subunit and thus inhibit DNA synthesis
2) specifically inhibit thioredoxin or thioredoxin reductase so that the SH groups of RR cannot be regenerated and again inhibit DNA synthesis
purpose of regulating ribonucleotide reductase?
to ensure balance in amounts between the pyrimidine bases
how is the ribonucleotide reductase reaction regulated by ATP?
[ATP] in cells is the highest of the nucleotides. it's a high energy signal, stimulates entire RR reaction by binding overall activity site.
since in high [ATP], ATP also binds spcificity site
ATP signal converts pyrimidines to deoxy-pyrimidines, so CDP -> dCDP and UP -> dUDP
how does regulation of the Ribonucleotide Reductase occur beyond the 1st step?
ATP signals to convert pyrimidines to de-oxypyrimidines
dCDP + dUDP eventually are metabolized to dCTP + dTTP by nucleotisde dipohsphate kianse
as dTTP builds up, it displaces ATP from substrate specificity site
dTTP specifies formation of dGDP, via GDP -> dGDP
dGDP -> dGTP, which slows down formation of deoxypyrimidines
once enough dGTP is produced, displaces dTTP from substrate specificity site and specifies ADP -> dADP
dADP -> dATP and dGTP production decreases
once enough dATP accumulates, it's a singal enough deoxynucleotides have been produced
dATP displaces ATP fomr overall activity site and shuts RR down
what is SCIDS?
what causes it?
severe combined immunodeficiency syndrome, aka bubble boy syndrome
due to deficiency in adenosine deaminase, which degrades ATP and dATP to hypoxanthine and xanthine
why does SCIDS occur re: its deficiency?
deficiency of enzyme that breaks down ATP or dATP to hypoxanthine or xanthine
this means do not have DNA synthesis
this means do not make antibodies
this means have no B and T cells, have low immune response, WBC don't function and divide properly, and get build up of dATP so inhibits ribonucleotide reductase
what are key diffs between CPS II and CPS I and what is significance of these diffs?
CPSI: urea cycle, gets N from NH4+, in the mito, activated by N-acetylglutamate - expulsion of NH3
CPS II: pyrimidine synthesis, gets N from Glutamine, actiavted by PRPP, inhibited by UTP - biosynthesis
how is synthesis of UMP regulated?
end product inhibited by its own present - it binds to CPS II
why does a defciency of ornithine transcarbamylase increase orotic acid levels?
if the carbamoyl phosphate made in the 1st step of the urea cycle isn't used due to a deficiency of this enzyme, then get build up of carbamoyl phosphate
if that isn't used in urea cycle, it'll be used in the pyrimidine synthesis pathway, resulting in the manufacture of a lot of orotic acid
this also would eliminate the normal feedback on this pathway via the UTP/UMP on CPS-II since CPS-I is making all of the carbamoyl phosphate in this scenario
why is deoxyribonucleotide ysnthesis decreased by free radical scavengers like hydroxy urea?
because free radical formation is critical to ribonucleotide reductase activity - so inhibiting free radcial fomaition would adversely impact synthesis of deoxyribonucleotides
how is overall activity of RR regulated
by whether ATP or dATP binds to overall activity site; ATP activates, dATP inhibits RR activity by binding there
how does dTTP affect substrat specificity of RR
as dTTP levels build up, it displaces ATP from substrate specificity site, specifies formation of dGDP via GDP -> dGDP which becomes dGTP
how are reduced thiols of RR regenerated?
by thioredoxin, which provides H's to reduce RR anew
Thioredoxin is reduced by Thioredoxin Reductase
Thioredoxin Reductase is reduced by FADH -> FAD
NADPH + H+ reduces FAD back to FADH, becomes NADP+
why does a deficiency of adenosine deaminase cause SCIDS?
beacuse lack of this enzyme means ATP and dATP can't be degraded to hypoxanthine and xanthine
this means that DNA synthesis cannot occur
this means antibody production is inhibited
this means the body lacks B and T cells, WBC don't function properly, there's a build up of dATP which inihibits ribonucleotide reductase activity