pyrimidine nucleotides

Question 1

pyrimidine rings

Answer 1

constituents of ring derived from glutamine (amide nitrogen from R-group), CO2 and aspartate (aspartic acid)

Question 2

steps in de novo synthesis of pyrimidine

Answer 2

1: carbamoyl phosphate synthetase II uses 2 ATP, CO2 and glutamine to make carbamoyl phosphate - releases glutamate, 2 ADP and Pi (regulated at this step)
2: aspartate transcarbamoylase converts carbamoyl phosphate to carbamoyl aspartate - asparated used, Pi released
3: dihydroorotase converts carbamoyl aspartate to dihydroorotate - uses H2O
4: dihydroorotate dehydrogenase converts dihydroorotate to orotate - uses NAD+ and releases NADH, H+
5: orotate phosphoribosyl-transferase adds ribose phosphate group to orotate (note that in pyrine synthesis, this group is there all along) - uses PRPP, releases PPi - makes orotidine 5’-monophosphate (OMP)
6: OMP decarboxylase makes uridine 5’-monophosphate (UMP) from OMP - releases CO2
7: series of nucleotide kinases converts UMP to UTP (analogous to synthesis of purine nucleotide triphosphates)
8: CTP synthase converts UTP to CTP - converts glutamine to glutamate and ATP to ADP + Pi in the process

Question 3

carbamoyl phosphate synthetase II (CPSII)

Answer 3

enzyme in first step of de novo pyrimidine synthesis
located in cytoplasm
uses 2 ATP, CO2, and glutamine to make carbamoyl phosphate
releases 2 ADP, Pi, and glutamate
inhibited by UTP, activated by ATP and PRPP in mammalian cells
regulated step in mammalian cells

note: this enzyme is not the same as carbamoyl synthetase I, which is mitochhondiral and part of the urea cycle

Question 4

aspartate transcarbamoylase

Answer 4

enzyme in second step of de novo pyrimidine synthesis
converts carbamoyl phosphate to carbamoyl aspartate
uses asparate
releases Pi
in prokaryotic cells, is site of regulation - inhibited by CTP

Question 5

dihydroorotase

Answer 5

enzyme responsible for third step of de novo pyrimidine synthesis
converts carbamoyl aspartate to dihydroorotate - uses H2O

Question 6

dihydroorotate dehydrogenase

Answer 6

enzyme responsible for fourth step of de novo pyrimidine synthesis
converts dihydroorotate to orotate
uses NAD+

Question 7

orotate phosphoribosyltransferase

Answer 7

enzyme responsible for fifth step of de novo pyrimidine synthesis
adds ribose phospate to orotate to make orotidine 5’-monophosphate (OMP)
uses PRPP
releases Pi

Question 8

OMP decarboxylase

Answer 8

enzyme responsible for sixth step of de novo pyrimidine synthesis
convert OMP to uridine 5’-monophosphate (UMP)
releases CO2

Question 9

CTP synthase

Answer 9

step 8 of de novo pyrimidine synthesis
converts UTP to CTP
uses glutamine - converts to glutamate
uses ATP - releases ADP and Pi

Question 10

salvage of pyrimidine bases

Answer 10

from diet and cell turnover
enzyme pyrimidine phosphoribosyltransferase converts
uses PRPP
not reversible
makes pyrimidine nucleoside monophosphate and PPi from pyrimidine and PRPP

Question 11

pyrimidine phosphoribosyltransferase

Answer 11

makes pyrimidine nucleoside monophosphate and PPi from pyrimidine and PRPP
responsible for salvage of pyrimidines

Question 12

degradation of pyrimidines

Answer 12

rings can be opened and degraded to soluble structures s.a. beta-alanine and beta-aminoisobutyrate - both can enter CA cycle

Question 13

ribonucleotide reductase

Answer 13

enzyme that converts ribonucleotide diphosphates (NDPs) to deoxyribonucleotide diphosphates (dNDPs) - so ADP, CDP, GDP, and UDP to dADP, dCDP, dGDP, and dUDP
multisubunited enzyme
releases H2O
uses thioredoxin as cofactor - oxidizes it - the thioredoxin shuttles NADPH to the active site of reductase - reductase uses reducing equivalent of NADPH
responsible for maintaining a balanced supply of dNTPs for DNA synthesis
expression level of enzyme regulated depending on cell’s needs
enzyme activity regulated by binding of nucleotides to allosteric sites
strongly inhibited by dATP

Question 14

adenosine deaminase (ADA) deficiency

Answer 14

if ADA deficient, conversion of adenosine to inosine (which can be made into urea to be excreted) is blocked => increase in cellular adenosine and dATP - dATP inhibits ribonucleotide reductase - can’t maintain dNTP pools
results in inhibition of ribonucleotide reductase and so some cells can’t proliferate
get SCID because T and B cells proliferation impaired by skewed dNTP pools

Question 15

synthesis of dTMP

Answer 15

ribonucleotide reductase makes dUTP but need dTMP for DNA synthesis
thymidylate synthase catalyzes conversion of dUMP to dTMP
gets methyl group from N5,N10-methylene-THF
THF oxidized to dihydrofolate (DHF)
cycle: DHF now converted to tetrahydrofolate by dehydrofolate reductase (using NADPH)
tetrahydrofolate now converted to N5,N10-methylene-tetrahydrofolate (uses serine, releases glycine)
N5,N10-methylene-THF used by thyrnidylate synthase to convert dUMP to dTMP, regenerating DHF
thymidylate synthase main target for chemotherapy

Question 16

thymidylate synthase

Answer 16

converts dUMP to dTMP
uses methyl group from N5,N10-methylene-THF
releases dihydrofolate (DHF)
target for drugs such as 5-fluorouracil (see future lectures for mechanism)

Question 17

dihydrofolate reductase

Answer 17

enzyme that converts dihydrofolate to tetrahydrofolate so it can then be converted to N5,N10-methylene-tetrahydrofolate
target for drug inhibition, eg methotrexate and aminopterin

Question 18

gout

Answer 18

due to excessive production or poor renal excretion of uric acid (end product of purine degradation)
can be diagnosed by presence of monosodium urate crystals in aspirated synovial fluid
attacks often happen at night because body temp down; when dehydrated (after sleeping);
uric acid not normally very soluble but can become more so if kidney can’t excrete it, body temp drops, in extremities (because cooler there), when dehydrated
most commonly because of failure to excrete but can also have overproduction

Question 19

causes of gout (3)

Answer 19

1: mutations in PRPP synthase that generate unregulated enzyme
2: lesch-nyhan syndrome, caused by loss of HGPRT involved in salvage of purine bases - de novo pathway driven forward (has elevated level of PRPP)
3: impaired kidney excretion or compromised kidney function

Question 20

allopurinol

Answer 20

drug used to treat gout
reduces uric acid synthesis at xanthine oxidase
inhibits de novo nucleotide pathway to prevent increased nucleotides

Question 21

nucleoside diphosphate kinase

Answer 21

XDP + ATP XTP + ADP
converts di-phosphates to tri-phosphorylated forms
needed because end-products of de novo pyrimidine and purine pathways are nucleoside monophosphates (XMPs) - these are converted to diphosphates by specific nucelotide monophosphate kinases and then to triphosphates (XTP) by nucleoside diphosphate kinases

Question 22

specific nucleoside monophosphate kinases

Answer 22

XMP + ATP XDP + ADP
end products of de novo pyrimidine and purine pathways are nuceloside monophosphates (XMP)
these enzymes convert the XMP to nucleoside diphosphates (XDP) so that these can then be converted to triphospates by nucleoside diphosphate kinase
example = uridine monophosphate kinase, that makes UDP from UMP

Question 23

synthesis of CTP

Answer 23

this is step 7 and 8 in the de novo synthesis of pyrimidines

1: UMP is phosphorylated by uridine monophosphate kinase to make UDP (note: the M means nonphosphate, the D means diphosphate)
2: nucleoside diphosphate kinase phosphorylates UDP to UTP (triphosphate = T)
3: CTP synthetase coverts UTP to CTP - uses glutamine and ATP - releases glutamate and ADP + Pi

Question 24

regulation of ribonucleotide reductase

Answer 24

expression level transcriptionally regulated, depending on cell’s needs - higher expression in rapidly dividing cells
balanced supply of dNTPs is maintained by complex allosteric regulation - depending on NT pools, enzyme displays preference for one substrate over another
imbalance in these pools can be mutagenic, results in DNA polymerase making too many mistakes
dATP inhibits enzyme activity (in ADA dAPT levels elevated)

Question 25

thioredoxin reductase

Answer 25

used to convert thioredoxin (S-S; oxidized form) back to thioredoxin (2 SH; reduced form) so that ribonucleotide reductase (which converts ADP, CDP, GDP, and UDP to deoxy forms) can use it again
uses NADPH, H+ and releases NADP+

Question 26

treatments for ADA

Answer 26

bubbles! isolate child
hematopoietic cell bone marrow transplantation from HLA-identical sibling
enzyme replacement therapy, some PEG-ADA taken up into plasma cells
gene replacement therapy: host T-cells isolated, transfected with retrovirus containing ADA encoding gene, infused back into individual