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amino acids are obtained from the diet when _______ are digested
________ ______ are degraded into amino acids because of
damage
misfolding
changing metabolic demands
proteins
cellular proteins
why are cellular proteins degraded?
misfolding
damage
changing metabolism demands
can excess amino acids be stored or excreted?
NO
dietary proteins are degraded to amino acids which are absorbed by _______?
what happens next?
intestine
one they are absorbed they are transported into bloodstream
how many essential amino acids are there that cannot be synthesized by the body and must be taken in through diet?
9
histidine
isoleucine
leucine
lysine
methione
phenylalanine
threonine
tryptophan
valine
the following is a list of
non essential amino acids
the digestion of dietary proteins begins in the _______ and is completed in the ______________
stomach —> intestine
what happens to dietary proteins in the stomach?
proteins are denatured into random coils
primary proteolytic enzyme of the stomach
at which pH is it most active?
pepsin
2
have all the dietary proteins converted into amino acids by the time they reach the intestine
NO
in the stomach, the proteins are partially degraded but not all are converted into amino acids yet
what is secreted as the partially degraded proteins from the stomach begin to enter the intestine (duodenum) ?
where are they secreted from?
sodium bicarbonate and proteolytic enzymes- PANCREAS
aminopeptidases - plasma membrane of intestinal cells
SEQ Dietary Protein → Amino Acid
consume protein
where and how are proteins initially uncoiled?
_______ break apart partially digested proteins into _______ _______, ________, and ________ in the lumen of the ______________
what is released when the proteins reach this organ? are they released by the organ itself?
there are 7 transporters that exist specific to a different group of amino acids on the membrane of which cells?
how are amino acids released into the blood from the cells?
in the stomach due to low pH maintained by pepsin
peptidases amino acids, tripeptides and dipeptides
sodium bicarbonate and proteolytic enzyme released by pancreas and aminopeptidase from the intestine
intestinal cells
Na+ transporter (Na+ come into intestinal cell and amino acid leaves)
how are amino acids released into the blood from the intestinal cells?
sodium antiporter
sodium into intestine
amino acid out of intestine
the degradation and resynthesis of proteins that takes place CONSTANTLY in cells
why is this process essential?
protein turnover
removes short-lived or damaged proteins
do all proteins have around the same half life?
NO
some last 11 minutes, others 3 months, and some are forever
what is the half life of
ornithine decarboxylase (catalyze synthesis of polyamines)
hemoglobin
crystallin (lens protein)
11 minutes
3 months
never
is mRNA or protein turnover faster?
mRNA turnover
a novel CRISPR cas9 based gene knockout therapy is in development with goal of decreasing protein C in liver
Liver levels will have to decrease by at least 75% for CRISPR to be effective
If protein X has half life of 1 day how long after succesful knowckout of Protein X ?
half life = 1 day so in one day 50% gone
after 2 days 50% of the remaining product will be gone
2 days
decreased by 75% by 2 half-lives 50% + 25%
___________: a small protein with _____ amino acids that tags proteins for destruction
what is special about this protein?
ubiquitin
76
ALL eukaryotes have them and they are highly conserved
Ubiquitin attaches by its ___________- terminal ____________ residue to the E-amino groups of 1+ _________ residues on the target protein
what is a requirement for this to take place?
what bond is formed and why?
carboxyl glycine
lysine on
require ATP hydrolysis
isopeptide bc/ e-amino groups are targeted
what bond forms between ubiquitin and its target protein and why?
isopeptide bond
targets has e-amino group not a-amino group (peptide bond)
does ubiquitin or its target protein have lysine residues ?
BOTH
ubiquitin has 7 lysine residues
Ubiquitin has an extended __________ terminus which is activated and linked to proteins targeted for destruction
Ubiquitin also has 7 ______ residues
carboxyl (C-terminus)
lysine
which three enzymes participate in the attachment of ubiquitin to a protein to be depredated
ubiquitin ACTIVATING enzyme (E1)
ubiquitin-CONJUGATING enzyme (E2)
ubiquitin- protein LIGASE (E3)
SEQ Ubiquitination
Using ATP, the ubiquitin- ________ enzyme (E1) adenylates ubiquitin and transfers a _______ group of a ____ residue to E1
Ubiquitin- __________ enzyme (E2) transfers ubiquitin to one of its own _________ groups
Ubiquitin protein ________ (E3) transfers ubiquitin from E2 to an ___-amino group on the TARGET PROTEIN
E3 brings E2 and the target protein together Ubiquitin can be transferred directly or be passed to a _____ residue of E3 first
activating sulfhydryl cys
conjugating sulfhydryl
ligase E
cys
which enzyme adenylates ubiquitin and transfers a sulfhydryl group on its cys residue?
Does this step require ATP?
E1 = ubiquitin activating enzyme
what is the role E2 in ubiquitination of target proteins?
E2 replaces E1 by transfers ubiquitin to one of its own sulfhydryl groups on its cys residue
what is the role of E3 in the ubiquitination of target proteins?
bring target cell and ubiquitin close together
transfers ubiquitin from E2 to e-group on TARGET protein
does each protein targeted for degradation have one ubiquitin protein bound?
NO has multiple ubiquitin bound
what are the two different ways a ubiquitin chain can be formed
E3 remains bound allowing for a chain of ubiquitin to form
E3 disassociates and an additional E2/E3 extends chain
how are ubiquitin molecules attached to one another in a chain?
a chain consisting of how many ubiquitin molecules are ideal for protein degradation?
ubiquitin binds to the N terminus or lysine residue of another ubiquitin
4+ ubiquitin linked by lys 48
how many diffferent genes are there for each enzyme?
E1
E2
E3
which provides the protein target specificity?
2
30-50
600
Since there are so few genes encoding for E1 and E2 many target proteins will interact with the same E1 and E2
however since there are 600 E3 genes, E3 can be specific in its target as the chances of having the same E3 is a lot less than E2 and E1
prevents random proteins that don’t need to be degraded to be tagged with ubiquitin
in _______________ 4 ubiquitin molecules are linked by ________ bonds
the ___- amino group of a _____residue of one ubiquitin is linked to the terminal CARBOXYLATE of another
tetraubiquitin
isopeptide
E lysine
this unit is the PRIMARY signal for degradation when linked to a target protein
tetraubiquitin
a specific sequence of amino acids that indicates a protein should be degraded
degron
for many target proteins the _______terminal residue amino acid (__-________) is an important degradation signal for E3 enzymes
may only be exposed after ________ cleavage
may be added after protein _________
may require other modifications such as ___-________ ________/______
amino
n-degron
proteolytic
synthesis
n-degron acetylation/ methylation
what are three examples of degrons?
n-degron
cyclin destruction boxes
PEST sequences
amino terminal residues can determine the half life of cytoplasmic yeast proteins
would a target protein with a n-terminal residue with a high affinity to E3 be considered stale or unstable?
Higher affinity to E = _______ half life
UNSTABLE bc/ higher chance of being ubiquinized
smaller
what would be the impact of not having enough E3?
what would be the impact of having overactive E3?
overall inappropriate turn over ?
underactive = protein accumulation of proteins that should have been targeted for destruction by E3
overactive = autism
inappropriate = cancer
ubiquitination regulates proteins involved in
______ repair
______ remodeling
_____ immunity
_________ ___________
___________
DNA
chromatin
innate
membrane trafficking
autophagy
Burkitt Lymphoma is driven by mutation to the onco-protein c-Myc.
what is likely to be true about c-Myc mutations in this cancer?
block ubiquitarian site on the protein
onco-proteins are able to rapidly divide because of the lack of degradation due to blocked ubiquitarian sites
what happens to proteins post ubiquitination?
ubiquitin tagged proteins are digested by proteosomes
a large, ATP-driven protease complex that digests ubiquinated proteins
Proteosome (26S proteosome)
what are the two subunits of 26S protease?
what is the difference between the two?
which is arranged as a barrel?
20S - does the actual catalyzing (looks like barrel)
19S - attracts the ubiquitin into 20S core
19S regulatory unit of 26S proteosome:
contains ubiquitin ___________ that bind specifically to ____________________ chains
uses _____ to unfold ____________ chains and direct them into the ______ core (20S )
contains _______________ to cleave off ubiquitin. why?
receptors POLYubiquitin
ATP POLYubiquitinated catalytic
isopeptidase cleaves ubiquitin so it can be reused in the future
key components of the 19S complex are ___ ATPases of the _____class
a class of _________ -like ATPases associated with the assembly, operation, and disassembly of protein complexes
6
AAA+
chaperone
the 20S proteasome is _______ shaped and made up of _____homologous subunits
subunits ___-type and ___-type are arranged in 4 rings of _______ each
some of the __-type subunits include protease active sites at their amino termini
barrel
28
a and b 7
b
The PROTEOLYTIC active sites of the _____ barrel:
three types of active sites in the __-subunits each with different specificity
what are the three different types of active sites within the barrel?
20S
beta
chymotrypsin, trypsin, and caspase-like
The _____________active sites of the 20S barrel:
All active sites employ N terminal ____ residue
the ______ group of the ____ residue attacks the ______ groups of peptide bonds, forming ACYL - ENZYME intermediates
substrates are degraded in a processive matter with/without intermediate release
substrates are reduced to peptides spanding from ___-____ residues before release
proteolytic
thr
hydroxyl thr carbonyl
WITHOUT
7-9
Proteosome —> Free Fatty Acid:
ubiquinated proteins are processed to _______ fragments
________ is removed and recycled PRIOR to protein degradation
_________ ___________ are further digested to yield free ______ _____________ which can be used for biosyntheic reactions such as _______ synthesis
OR
amino group can be removed and processed to ______ and the carbon skeleton can be used to synthesize __________ or ______ or used directly as fuel for cellular respiration
peptide
ubiquitin
peptide fragments amino acids PROTEIN
UREA carbohydrates or fats
what are the 4 different paths a ubiquitinated protein can take once they are degraded into amino acids in the 20S subunit of 26S proteasome?
amino acids —> proteins
amino acid—> remove amino group —> urea —> carbon skeleton used for CARBOHYDRATES
amino acid —> remove amino group—> carbon skeleton used for FATS
used for fuel for CELLULAR RESPIRATION
gene transcription
cell-cycle progression
circadian rhythms
organ formation
inflammatory response
tumor suppression
cholesterol metabolism
antigen processing
The following are ALL controlled by which pathway?
ubiquitin - proteosome pathway
a dipeptidyl boronic acid inhibitor of proteasome
used in multiple myeloma
increase proteins by decreasing degradation by proteosome
bortezomib (Velcade)
used as regulatory mechanisms for protein expression
degrons
why is bortezomib effective multiple myeloma
by inhibiting proteosomes bortezomib increases the level of pro-apoptotic protein factors
** don’t confuse yourself pro-apoptotic factors are not proteosme but other proteins!**
suicide (irreversible) inhbiitor of the proteosome of M. tuberculosis (no effect on human proteosomes)
HT1171
amino acids are NOT able to be excreted as they are
the first step in amino acid degradation is the removal of __________
the remaining carbon skeleton is _________ to a _______ ________ or to acetyl CoA
where is the major site of amino acid degradation?
nitrogen
metabolized glycolytic intermediate
liver + muscles
a-amino group is transferred to a-____________ yielding _______
_________ is then __________ __________ to yield ammonium ion ( NH4+)
ketoglutarate
glutamate
glutamate oxidatively deaminated
which enzyme catalyzes the transfer of an a-amino group from an a-amino acid to an a-ketoacid
aminotransferase
__________ __________ and ___________ ______________ catalyzes the transfer of the amino group aspartate to a-ketoglutarate
aspartate aminotransferase and alanine aminotransferase
aspartarte + a-ketoglutarate ↔ oxaloacetate + glutamate
aspartate amino transferase
aspartate amino transferase catalyze the transfer of the ________ ______ of aspartarte to a ______________
amino group
a-ketoglutarate
alanine aminotransferase catalyzes the transfer of the ______ ______ of alanine to ___- _________
amino group a-ketoglutarate
what are the products of transferring an a-amino group of alanine to a-ketoglutarate using alanine aminotransferase?
glutamate + pyruvate
what are the products of transferring an a-amino group from an aspartate onto an a-ketoglutarate?
glutamate + oxaloacetate
the presence of _________ and _________ in the blood is an indication of liver damage
alanine amino transferase
and
aspartate amino transferase
liver damage can occur due to
in cases of liver damage, liver cell _______ are damaged and ___________ leak into the blood
viral hepatitis
long-term excessive alcohol consumption
reaction to drugs (acetaminophen)
membranes aspartate and alanine aminotransferase
aspartate and alanine aminotransferases require _______ ______ (PLP) which is derived from vitamin ____
pyridoxal phosphate
B6
Step 1 of transamination is to transfer the amino group from the amino acid substrate to ______ (coenzyme) and release of _________
Step 2 of transamination is to transfer the amino group from ______ onto the ________ to generate a new _______ _________
PLP
ketoacid (a- ketoglutarate)
PLP ketoacid (a- ketoglutamate) amino acid (pyruvate or oxaloacetate)
_______ _______ enzymes catalyze a wide array of reactions at the ____- carbon of amino acids
including
deaminations
racimizations
decarboxylation
adol cleavages
pyridoxal phosphate alpha
_______ _______ enzymes catalyze a wide array of reactions at the ____ and ____carbon of amino acids
including
elimination
replacement
pyridoxal phosphate
B and Y
a mitochondrial enzyme that converts the
nitrogen atom from glutamate —> ammonia (NH3) + a-ketoglutarate
through OXIDATIVE DEAMINATION
this enzyme is found in the ________ and uses _____ or _____ for energy
glutamate dehydrogenase
liver
NAD+ or NADP+
glutamate dehydrogenase converts glutamate to a-ketoglutarate and a free ammonia ion (NH3) using oxidative deamination
how exactly does this work?
the C—N bond is ___________
hydrolysis of __________ intermediate
this process is inhibited by _______ and stimulated by ________ in mammals
dehydrated
ketamine
GTP ADP
serine and threonine can be directly deaminated by _____ ______ and ________ _______
PLP acts as a ___________ group (coenzyme)
NO transfer of the a- amino group to ____________ from _____is required
_________ precedes deamination
serine dehydratase
threonine dehydratase
prosthetic
a-ketoglutarate PLP
dehydration
which enzyme allows this reaction to take place without needing PLP to obtain the amino group first and then transfer it onto the a-ketoglutarate?
glutamate dehydrogenase
what is the ketoacid that gains the amino group and the amino acid product of
alanine transamination
aspartate transamination
alanine
ketoacid = a-ketoglutarate
amino acid = pyruvate
aspartate
ketoacid = a-ketoglutarate
amino acid = oxaloacetate
Serine —> ____________ —> NH4+
Threonine —> _____________ —> NH4+
dehydration completed with serine and threonine dehydratase without needing PLP coenzyme
pyruvate
a-ketobutyrate
in most terrestrial vertebrates, NH4+ is converted into ______
the sume of the reactions of aminotransferase and glutamate dehydrogenase is
__-
what happens to that molecule?
urea and it is excreted
the sum of the reactions of aminotransferases and glutamate dehydrogenase is
a-amino acid + NAD+ + _____ ↔
a-ketoacid + NH4+ +NADH (NADPH) +____
H20
H+
Peripheral tissues transport _______ to the liver
muscles use branched _____ _______as fuel during prolonged excersize and fasting
______ is transported from muscle to the liver as ______ (through ______) in the glucose-_______ cycle
nitrogen
amino acids
nitrogen
nitrogen alanine (glutamate) alanine
glutamaglutAMINE SYNTHETASE catalyzes the synthesis of glutamine from _________ and ____
the nitrogen of glutamine can be eliminated by incorporation into ______ in the liver
____ + ____ + ______ *glutamine synthetase*—>
glutamine + ____+ ______
NH4+ and glutaMATE
UREA
NH4+ + glutamate + ATP
Pi + ADP
Glucose- Alanine Cycle:
during prolonged exercise and fasting, muscle uses branched-chain amino acids as fuel
the nitrogen removed from the amino acid is transferred (through _______) to ________ which is released into the BLOODstream
in the liver ________ is taken up and converted into _______ for the synthesis of glucose
glutamate alanine
alanine
alanine pyruvate
The urea cycle eliminates both nitrogen and carbon waste products:
how many nitrogen enter the cycle and leave as urea?
what else is eliminated simultaneously as it it is hydrated to bicarbonate which enters the cycle ?
_______ ______ _______ I catalyzes the coupling of ammonia (NH3) with bicarbonate (HCO3-) to form ___________ ________ in the mitochondria
mammals have 2 isozymes
requires 2 molecules of _____ making the reaction IRREVERSIBLE
2
carbon dioxide
carbamoyl phosphate synthetase
carbamoyl phosphate
ATP
_______ ______ _________ is the KEY regulatory enzyme for urea synthesis
requires allosteric regulator__ -_____ for activity
is __________ by acetylation and __________by deacetylation
when is this allosteric regulator activated?
carbamoyl phosphate synthetase I
N-acetylglutamate
inhibited stimulated
when amino acids are HIGH
when is N-acytelglutamate synthesized?
which enzyme catalyzes its synthesis
when there is ample amino acids
N-acytelglutamate synthase
Carbamoyl Phosphate reacts with ________ to begin the urea cycle
_________ ____________ catalyzes the transfer of the carbamoyl group of the carbamyl phosphate to _________ forming CITRULLINE (which is transported into the cytoplasm from the mitochondria)
ornithine
ornitine transcarbamoylase ORNITHINE
_________ is the SECOND donor of the nitrogen used to make urea
where does the first nitrogen come from?
aspartate
first: NH4+ and bicarbonate come together to form carbamoyl phosphate
UREA Cycle: which steps occur in the mitochondria and which occur in the cytoplasm?
NH4+ + HCO3- → carbomyl phosphate
carbomyl phosphate + ornithine —> citrulline
citrulline + aspartate —> arginine succinate
arginine succinate—> arginine + fumarate
arginine —> UREA +
first two steps in mitochondria
rest in CYTOPLASM starting from aspartate + citrulline —> arginine succinate
UREA CYCLE: which enzymes catalyze each step?
NH4+ + HCO3- ———→ carbomyl phosphate
carbomyl phosphate + ornithine ——> citrulline
citruline + aspartarte —> arginino succinate
arginino succinate —> Arginine + Fumarate
Arginine —> UREA + ornithine
carbomyl phosphate synthetase I
N-acytelglutamate
N-acytelglutamate synthetase (if high amino acid)
orthinine transcarbomylase
arginosuccinate synthetase + ATP
argininosuccinase (cleaves)
arginase (cleaves)
Urea Cycle:
NH4+ + HCO3- ———→ carbomyl phosphate
carbomyl phosphate + ornithine ——> cirtrulline
what does cirtruline combine with and what does it form?
which enzyme is used?
citruline + aspartarte —→ arginosuccinate
arginosuccinate synthetase
Urea Cycle:
NH4+ + HCO3- ———→ carbomyl phosphate
carbomyl phosphate + ornithine ——> cirtrulline
citrulline + aspartarte —> aspartate succinate
what happens to aspartate succinate? what are the next products that are formed?
aspartarte succinate is cleaved by argininosuccinace giving
arginine and fumarate
UREA CYCLE:
NH4+ HCO3- (bicarbonate) —> carbomyl phosphate
Carbomyl Phosphate + Urothione —> Citruline
Citruline + Aspartarte —> aspartarte succinate
once aspartarte succinate is cleaved to arginine + fumarate what happens to each?
arginine cleaved to generate urea and ornithine
ornithine is trasnfered back into the mitochondria
urea is excreted
what are we left with once arginine is cleaved by arginase?
what happens to each of those products?
urea (excreted)
ornithine (transported back to mitochondria to react with carbomyl phosphate to form citrulline )
the urea cycle is linked to _________
the stoichiometry of the urea cycle is:
CO2+ NH4+ + aspartate + 3 ____+ 2 ____ —>
Urea +fumurate + Pi + PPi+ ____+ 2 _____
fumarate is HDYRATED to maleate which is OXIDIZED to _______ which can be converted into GLUCOSE
gluconeogenesis
3 ATP +2H2O
2ADP+ AMP
oxaloacetate
what does the muscle lack causing Nitrogens to be sent to the liver as alanine (through glutamate) in the glucose-alanine cycle.
enzymes for urea synthesis
oxaloacetate can be formed from which product of the urea cycle?
which reactions does the product have to go through to become oxaloacetate?
what two different things can then be done with the oxaloacetate?
fumarate
fumarate can be dehydrated to maleate then oxidized to oxaloacetate
gluconeogenesis form glucose from oxaloacetate
transamination to aspartarte to donate its N and combine with citrulline to create urea to be excreted
the urea cycle, gluconeogenesis, and the transamination of oxaloacetate are linked by _______ and _________
fumarate and aspartate
what can cause an increased level of NH4+ (hyperammonemia) in the blood and later brain damage n patients?
defect in urea cycle!
NH4+ not combining wiith HCO3- to make cabomyl phosphate and ultimately get rid of NH4+ as urea
high levels of NH4+ due to defects in urea cycle can lead to
inappropriate activation of _____, ____, ____ cotransporters disrupting OSMOTIC BALANCE of the nerve cell causing cellular ______
disrupt _________ systems
impact energy ________, levels of _____ stress, _____ oxide synthesis, and signal transduction pathways
Na+, K+, Cl- swelling
neurotransmittter
metabolism oxidative nitric
an arginosuccinase deficiency can be managed by
increasing / decreasing total protein intake
supplementing _______ in diet
excess ________ is excreted in the form of arginosuccinate
DECREASING
arginine