biochem lecture 11 pt1 Flashcards
what are the two biosynthesis pathways for nucleotides
de novo synthesis, salvage pathway
what are we focusing on
biosynthesis of the nucleotides, building blocks for DNA/RNA (not actual DNA synthesis)
what is de novo synthesis
compounds synthesized from scratch/minimal components
what are building blocks for de novo synthesis of nucleotides
metabolic precursors; parts of AAs, ribose-5-phosphate (byproduct of pentose phosphate pathway),
what is ribose-5-phosphate important for
providing the structure that will become either the deoxyribose or ribose sugar in DNA and RNA
what are salvage pathways
synthesis of nucleotides from scavenged components
what are scavenged components
recycled free bases, nucleosides (present from breakdown of nucleic acids)
what are 2 types of nitrogen containing bases
purines and pyrimidines
describe purines structure
bicyclic
describe pyrimidine structure
single ring
what are the purines
adenine, guanine
what are the pyrimidines
uracil, thymine, cytosine
for purine de novo biosynthesis, what are the sources of parts of purine bicyclic ring structure
aspartic acid, formate, glycine, amine N of glutamine, Co2
what is important precursor for purine de novo synthesis
5-phosphoribosyl 1-pyrophosphate (PRPP)
what does de-novo synthesis of purine begin and end with
begins with PRPP, ends w/ inosinate (IMP) formation
what is another important intermediate
inosinate (IMP) or inosinate monophosphate
why is IMP formation important
from IMP synthesis, we have a bifurcation
what is the purine bicyclic ring structure built off of in de novo synthesis
PRPP; serves as precursor for sugar
what is IMP an important common intermediate for
synthesis of both AMP and GMP
what do we have in each of these split, bifurcated pathways
an E source (GTP or ATP), and an amino group source (NH3)
what do we have as the E source for AMP synthesis
GTP
what do we have as the amino group for AMP synthesis
aspartate
describe what’s in AMP synthesis
GTP as E source, aspartate provides amino group that’s part of adenylate structure for AMP
what is E source for GMP synthesis
ATP
what is amino group source for GMP synthesis
glutamine
describe what’s in GMP synthesis
ATP as an E source, glutamine as an amino source
what are both AMP and GMP
parent compounds
what happens to AMP and GMP
go through a set of phosphorylated rxns that will result in triphosphorylated forms
describe regulation of adenine and guanine nucleotide synthesis in bacteria
3 major feedback inhibition mechanisms/points of control
what are the 3 points of control in adenine/guanine nucleotide biosynthesis
PRPP syntethase, glutamine-PRPP amidotransferase, and adenylosuccinate synthesis
what do the 3 points of control represent
the 3 major feedback inhibition points
how do we balance the pool of different nucleotides in terms of ensuring you have appropriate levels of all 4/5 diff nucleotides
coordinated feedback inhibition
what does pyrimidine de novo synthesis involve
aspartate, PRPP, carbamoyl phosphate
what is the first enzyme in pyrimidine de novo synthesis pathway
aspartate transcarbamoylase or ATCase
how does purine pathway work
start off w/ PRPP, construct nitrogen containing base off the ribose moiety of that structure
how does pyrimidine pathway work
construct monocyclic ring first, middle of the pathway we attach PRPP or what eventually becomes ribose sugar
is there a difference between the purine and pyrimidine biosynthesis pathways
yup
what are the basic precursors for pyrimidine biosynthesis pathway
aspartate, PRPP, carbamoyl phosphate
basically how does pyrimidine biosynthesis work
we have the 6 member pyrimidine ring made first, that’s gonna be attached to ribose-5-phosphate
what carbamoyl phosphate
intermediate used in first step of pyrimidine de novo pathway
what is important for synthesis of carbamoyl phosphate
bacterial carbamoyl phosphate synthetase
what does bacterial carbamoyl phosphate synthetase use in synthesis of carbamoyl phosphate
glutamine and ADP
describe bacterial carbamoyl phosphate synthetase
multi-subunit enzyme complex
what does bacterial carbamoyl phosphate synthetase use
substrate channeling
what is substrate channeling
helps retain/channel/keep intermediates that are part of synthesis step close to the enzyme, so that we don’t lose those intermediates to diffusion
what happens if we lose intermediates to diffusion
can lessen efficiency of enzyme
when is substrate channeling particularly important
if intermediates are short-lived; if they decay or are unstable, we can lose those intermediates before they’re utilized in this enzyme
how many active sites in bacterial carbamoyl phosphate synthetase
3 separate active sites
is ATCase same as the carbamoyl phosphate synthetase
no; that makes carbamoyl phosphate, so it’s made by the time we get to first step in pyrimidine pathway
what is the first enzyme in the pyrimidine de novo synthesis pathway
ATCase (aspartate transcarbamoylase)
what kind of enzyme is ATCase
allosterically regulated enzyme
how do we know ATCase is allosterically regulated
because of the sigmoidal shaped curve
what is an important regulator of ATCase
CTP (cytosine triphosphate)
what does ATP do
reverses effects of CTP; serves as an antagonist, restores the same level of activity
what is a rightward shift indicative of
something inhibiting the enzyme (takes more of substrate to reach the same V)
what is CTP
feedback inhibitor of ATCase
what else is CTP
end product of one of the pyrimidines that are synthesized, so it serves as a feedback inhibitor
describe feedback inhibitory mechanisms in synthesis pathways
with synthesis pathways we’re utilizing energy that’s precious to the cell. when we have enough of end product, there are feedback inhibitory mechanisms that dampen/slow down/inhibit that process
what does ATP do
antagonistic effect on CTP; restores normal activity of ATCase
what happens if there’s an imbalance of pyrimidine vs. purine pool and there’s more ATP
ATP is purine, pyrimidines need to be balanced w/ the purine concentration in the cell, so more pyrimidines
what does the first enzyme in the pathway of pyrimidine de novo synthesis do
serves as point of regulation
what does CTP at the bottom do
serves as allosteric inhibitor (negative allosteric regulation of aspartate transcarbamylase)
what are 2 general types of nucleotide biosynthesis
ribonucleotides (RNA precursors), deoxyribonucleotides (DNA precursors)
what do ribonucelotides serve as
precursors of deoxyribonucleotides
what is made first in the de novo pathway
ribonucleotides
what enzyme carries out the series of redox reactions to go from ribose to deoxyribose
ribonucleotide reductase complex
what is deoxyribose to ribose
reduced form of ribose sugar
what does ribonucleotide reductase act on
ribonucleotide diphosphates (diphosphorylated forms of ribonucleotides)
what is ribonucleotide diphosphate being converted to
deoxyribonucleotide diphosphate
what does this mechanism culminate in
synthesis of dNDP or deoxy form of nucleotide diphosphate
basically what are we goin gfrom
OH group at the C2 carbon to H at that carbon
what serves as source of electrons
NADPH
what two pathways are important
one for transfer of electrons to NDP substrate, but also a resetting of the ribonucleotide reductase
what does ribonucleotide reductase have
two sulfhydryl groups, which correspond to specific cysteine residues within active site of enzyme
what is needed for enzyme to function
needs to be reset into the reduced form
what happens in the last redox step
convert NDP to dNDP, the thiol/sulfhydryl (SH) groups get oxidized and form a disulfide bridge (S-S)
describe path electrons take
NADPH to FAD, FAD to thioredoxin, ultimately electrons get transferred to ribonucleotide reductase
how does all this get set into motion
we’re re-reducing these sulfurs that are part of the system
how do we re-reduce the sulfurs
NADPH (serves as feeder source of electrons), sequential redox, GSH (glutathione), so we have glutathione reductase. from glutathione reductase electrons are transferred to another protein glutaredoxin. from glutaredoxin we transfer electrons to ribonucleotide reductase to regenerate the reduce form of reductase (SH groups instead of S-S bridge)
what is this ribonucleotide reductase complex similar to
pyruvate dehydrogenase complex (idea of having to reset the system thru redox)
what do we need to do in order to allow for multiple rounds of reduction of NDP to dNDP
we have to regenerate these reduced thiol (SH) groups for each cycle
what do we end up doing to the sugar
replace 2’ OH group w/ 2’ H
do we deplete all of the ribose (turn it all into deoxyribose?0
no, we use some of that to make building blocks for RNA. but others will be used for generating deoxyribose form of sugar (DNA synthesis)
what kind of regulation does ribonucleotide reductase have
a complex kind of allosteric regulation
how many levels of control in ribonucleotide reductase
2 levels of control
what are the two levels of control in ribonucleotide reductase
primary regulation site, substrate specificity site
what does primary regulation site serve as
basic on-off switch for enzyme
how many regulatory subunits are there
2; R1 and R1
how many catalytic subunits are there
1; R2
what does active site have
thiols/cysteines with SH groups, where substrates bind
what is the point of having a combined primary regulatory site (on-off switch) and substrate specificity site
to provide balanced concentration of all the diff types of nucleotides or deoxynucleotides
what do we want in terms of the diff nucleotides
don’t want to be able to carry out the reduction of just one type of ribonucleotide diphosphate; we want all the diff ones (A, T, Cs, Gs) to be reduced so that we have a balanced pool of those diff building blocks
what happens when we have an abundance of one type of nucleotide
effectively stimulates production of other types of nucleotides
what is the general idea behind this regulation
to have balanced pool of all the diff nucleotides
what is ribonucleotide reductase regulated by
dNTPs
why does dNTP serve as a feedback inhibitor of ribonucleotide reductase
this enzyme converts ribonucleotide diphosphates into deoxyribonucleotide diphosphates, when we have subsequent phosphorylation of those di-phosphorylated forms to generate triphosphorylated forms, when there’s enough of the deoxy form of ATP, it will serve as a feedback inhibitor of enzyme
what serves as an activator for ribonucleotide reductase
having more of the ribonucleotide (ribo ATP form)
what effects do ATP vs dATP have on enzyme activity
opposing
what is the rule regarding substrate specificity
the nucleotide that serves as an allosteric regulator of substrate specificity is gonna favor reduction of other or alternate types of nucleotide diphosphates
what do dATP or ATP favor
reduction of UDP and CDP
what do dTTP or dGTP favor
reduction of GDP and ADP
what happens after we convert the ribo NDP to deoxy ribo NDP form by reductase
we need to go through additional phosphorylation
what generates the tri-phosphorylated forms
a series of kinases
what converts dCDP into dCTP
nucleoside diphosphate kinase
what happens to dUDP
converted to dUTP by the same enzyme
describe CTP and UTP
same thing, except CTP has an amino group constituent off the ring structure
what has to happen to go from dCTP to dUTP
deamination reaction, by enzyme deaminase
what does deamination reaction do
converts cytosine base into uracil base
what does dUTPase do
converts dUTP into dUMP
what is dUMP
important precursor for eventual synthesis of dTMP
what are the triphosphorylated forms of these nucleotides
what is used as eventual precursors for DNA and RNA synthesis
what are major products generated in degradation of purines and pyrimidines
uric acid and urea
what is an end product for degradation of GMP and AMP
uric acid
what do primates/mammals excrete
excess nitrogen
is nitrogen useful from an energetic standpoint
not really
what do primates generate from degradation of purines and pyrimidines
uric acid
but what do primates excrete the bulk of their nitrogen as
urea in urea cycle (as opposed to uric acid in purine degradation)
what are the two sources of excreted nitrogen
one from purines and pyrimidines, other from proteins and AAs that are degraded & used that pass thru urea cycle
do we excrete urea or uric acid
some uric acid, but majority of excreted nitrogen is in form of urea
who excretes uric acid
primates, birds, reptiles, insects
who excretes allantoin
most mammals
who excretes allantoate
bony fishes
who excretes urea
amphibians, cartilaginous fishes
who excretes ammonia
marine invertebrates
what is most abundant form of excreted nitrogen in primates, and where does that come from
urea; comes from degradation of proteins and AAs
important intermediate in catabolism of pyrimidines
methylmalonyl semialdehyde
what is methylmalonyl semi aldehyde degraded to
succinyl CoA (TCA cycle intermediate)
what is succinyl CoA
TCA cycle intermediate
what happens to carbon skeleton derived from catabolism of pyrimidines
can be shunted into TCA cycle
what are salvage pathways
pathways that scavenge or utilize free purine pyrimidine bases
how are these free bases released
thru degradation of nucleotides
do all nucleotides go through degradative pathways
not all, some can be salvaged and reused
how are free purine and pyrimidine bases released
via metabolic degradation of NTs
what are free purines used
salvaged and reused to make NTs
what is a major pathway for purines
adenine + PRPP –> generates AMP and PPi (pyrophosphate)
where else are there similar pathways
for pyrimidines in bacteria and mammals
what is a genetic disorder associated w/ defects in the salvage pathway enzyme
Lesch-Nyhan syndrome
Lesch-Nyhan syndrome is a result in defect in what enzyme
hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
what enzyme is HGPRT
salvage patwhay enzyme
what is Lesch-Nyhan syndrome
almost exclusive to male children; profound mental retardation, self-mutilating behavior
what is seen in Lesch-Nyhan synddrom
elevated levels of de novo purine synthesis, so increase in uric acid
what other conditions are there
gout
how does gout arise
excess production of uric acid
what is gout
painful disease, affects joints and other tissues
why is gout created
b/c there are certain competitive inhibitors
what are competitive inhibitors
compounds/analogs, structurally similar to naturally occurring precursor/intermediate/end product
what are the competitive inhibitors in gout
enzyme xanthine oxidase, oxypurinol is its competitive inhibitor
what is allopurinol
inhibitor of one of the enzymes that leads to production of excess uric acid
what is allopurinol an inhibitor of
xanthine oxidase
what is oxypurinol
byproduct of action of xanthine oxidase on allopurinol drug; serves as competitive inhibitor of xanthine oxidase
what does oxypurinol do
inhibits XO
where else do we see structural analogs
in drugs, chemo
what is a common target of these chemotherapeutics
to target enzymes part of nucleotide biosynthesis pathway
why does it make sense that chemo targets these enzymes
tumor cells are v active, high rates of replication. one way to slow rate of proliferation is to block their ability to synthesize nucleotides, DNA and RNA, and precursors
what are some drugs designed for
to target diff points of nucleotide metabolism
what is one way to slow replication of tumor cells
block their ability to synthesize nucleotides, synthesize DNA/RNA, synthesis of precursors
what are two target points for nucleotide metabolism
enzyme thymidylate synthase, other enzyme is dihydrofolate reductase (DHFR)
what is one drug that targets these points
FdUMP
what is FdUMP
fluorine analog of the normal substrate for thymidylate synthase (dUMP)
what does thymidylate synthase do
converts dUMP to dTMP
what can happen to dTMP
become phosphorylated to generate dTTP, which is then used in DNA synthesis
what is another enzyme that is targeted by drugs
dihydrofolate reductase
what is dihydrofolate reductase
reductase; uses NADPH as an electron source, reduces folic acid
what is folic acid
an important precursor for certain nucleotides
what drugs target dihydrofolate reductase
methotrexate, aminopterin, trimethoprim
what are these drugs used for
to treat various types of cancers
what is FdUMP used for
to treat diff cancers
what does FdUMP serve as
suicide inhibitors
what are regular inhibitors
interact w/ enzyme via non-covalent interaction, binds and inhibits it. reversibly binding inhibitors; binding is reversible and can dissociate again
what are suicide inhibitors
utilized in the catalytic activity of the enzyme. rather than inhibitor being converted into product, it becomes covalently attached to the enzyme within its active site, forms this dead-end complex.
why is this dead-end covalent complex achieved
because there is a chemically reactive fluorine
what happens in normal conversion of dUMP to dTMP
normal 3 step process, produces dTMP at the end
what is FdUMP similar to
dUMP natural substrate for thymidylate synthase
what does FDUMP go thru
regular steps of catalysis, but reactive fluorine results in formation of this covalent dead-end complex
what happens after covalent dead-end complex
enzyme can no longer catalyze any reactions, because drug is covalently attached to the active site of the enzyme, so the enzyme is basically dead
why are they called suicide inhibitors
get used in the biochemical rxn catalyzed by the enzyme, that effectively results in an inactivated enzyme
