SYLLABUS 19: Nucleotide & Deoxynucleotide Metabolism

Question 1

what is the first pyrimidine made

Answer 1

UMP

UMP -> UDP -> UTP -> CTP via carbamyl phosphate

Question 2

what is CPS-II

1. where is it located
2. what inhibits it
3. what activates it
4. what is its source of N

Answer 2

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

Question 3

first step of pyrimidine synthesis?

Answer 3

CPS-II catalyzes reaction

glutamine + CO2 + ATP -> carbamoyl phosphate + glutamate

UTP or UMP end-product inhibits this rxn; PRPP activates it

Question 4

what inhibits or activates the 1st enzyme of pyrimidine synthesis?

Answer 4

CPS-II

PRPP activates it

UTP inhibits it

Question 5

what is 2nd step of pyrimidine synthesis?

Answer 5

rate limiting step: aspartate transcarbamoylase does reaction:

carbamoyl phosphate + aspartate -> N-carbamoylaspartate

**inhibited **by production of **CTP, **stimulated **by ATP

Question 6

what is rate limiting enzyme of pyrimidine synthesis?

what inhibits or stimulates it?

Answer 6

aspartate transcarbamylase

2nd step of the synthesis pathway

end-product inhibited by CTP, stimulated by ATP

Question 7

what is orotate?

Answer 7

an intermediate in the pyrimidine synthesis pathway

the most abundant deficiency in urea cycle = ornithine transcarbamylase, causes high levels of orotate in the blood

Question 8

what might cause high levels of orotate in the blood? why?

Answer 8

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

Question 9

how are nucleoside mono/di/tri phosphates interconvertable?

Answer 9

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

nucleoside diphosphates:

GDP + ATP <-> GTP + ADP or CDP + ATP <-> CTP + ADP

Question 10

how can a deoxynucleotide be converted to a ribonucleotide?

Answer 10

it’s not possible to convert a deoxynucleotide -> ribonucleotide but a ribonucleotide can -> deoxynucleotide by ribonucleotide reductase

Question 11

function of ribunucleotide reductase?

Answer 11

catalyzes converstion of ribose to deoxyribose base for DNA

Question 12

structure of ribunucleotide reductase?

Answer 12

2 major subunits, B1 and B2

B1 has:

activity site &

substrate specificity site &

substrate binding site

Question 13

function of the overall activity site?

what can bind to it, what’s the effect?

Answer 13

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

Question 14

what controls activity of the ribunucleotide reductase enzyme? how?

Answer 14

ATP binds activity site -> sitmulates synthesis of deoxyribonucleotides

dATP binds activity site -> prevents more synthesis of deoxynucleotides

Question 15

what function of substrate specificity site?

Answer 15

what binds here determines which ribonucleotide diphosphate will be converted to the deoxyribonucleotide diphosphate at the enzyme’s activity site

Question 16

function of substrate binding site?

Answer 16

where substrate binds

substrate is ribonucleotide diphosphate (XDP) which through the RR enzyme becoase doxyribunucleotide diphosphate (dXDP)

Question 17

what’s the interplay between purines and pyrimidines in the RR activation?

Answer 17

if ATP, a purine, binds to the substrate specificity site, it activates RR so that it converts pyrimidine substrates to deoxypyrimidines

Question 18

structure of B2 subunit of ribonucleotide reductase?

Answer 18

has iron oxygen catalytic centers

has a Y* radical that’s formed in the catalytic mechanism

Question 19

how does the ribonucleotide reductase system work?

Answer 19

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

Question 20

is H from the C3 ever lost in the RR reaction?

Answer 20

no

Question 21

what’s the function of thioredoxin and thioredoxin reductase?

Answer 21

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

Question 22

how could chemotherapy work with the RR system of enzymes?

Answer 22

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

Question 23

purpose of regulating ribonucleotide reductase?

Answer 23

to ensure balance in amounts between the pyrimidine bases

Question 24

how is the ribonucleotide reductase reaction regulated by ATP?

Answer 24

allosteric regulation:

[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

Question 25

how does regulation of the Ribonucleotide Reductase occur beyond the 1st step?

Answer 25

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

Question 26

what is SCIDS? what causes it?

Answer 26

severe combined immunodeficiency syndrome, aka bubble boy syndrome due to deficiency in adenosine deaminase, which degrades ATP and dATP to hypoxanthine and xanthine

Question 27

why does SCIDS occur re: its deficiency?

Answer 27

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 **

Question 28

what are key diffs between CPS II and CPS I and what is significance of these diffs?

Answer 28

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

Question 29

how is synthesis of UMP regulated?

Answer 29

end product inhibited by its own present - it binds to CPS II

Question 30

why does a defciency of ornithine transcarbamylase increase orotic acid levels?

Answer 30

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

Question 31

why is deoxyribonucleotide ysnthesis decreased by free radical scavengers like hydroxy urea?

Answer 31

because free radical formation is critical to ribonucleotide reductase activity - so inhibiting free radcial fomaition would adversely impact synthesis of deoxyribonucleotides

Question 32

how is overall activity of RR regulated

Answer 32

by whether ATP or dATP binds to overall activity site; ATP activates, dATP inhibits RR activity by binding there

Question 33

how does dTTP affect substrat specificity of RR

Answer 33

as dTTP levels build up, it displaces ATP from substrate specificity site, specifies formation of dGDP via GDP -\> dGDP which becomes dGTP

Question 34

how are reduced thiols of RR regenerated?

Answer 34

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+

Question 35

why does a deficiency of adenosine deaminase cause SCIDS?

Answer 35

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