4: Protein Turnover and Amino Acid Catabolism

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?

1. misfolding

2. damage

3. changing metabolism demands

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?

1. misfolding

2. damage

3. 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

1. histidine

2. isoleucine

3. leucine

4. lysine

5. methione

6. phenylalanine

7. threonine

8. tryptophan

9. 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

1. consume protein

2. where and how are proteins initially uncoiled?

3. _______ 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?

4. there are 7 transporters that exist specific to a different group of amino acids on the membrane of which cells?

5. how are amino acids released into the blood from the cells?

2. in the stomach due to low pH maintained by pepsin

3. peptidases amino acids, tripeptides and dipeptides

   • sodium bicarbonate and proteolytic enzyme released by pancreas and aminopeptidase from the intestine

4. intestinal cells

5. 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

1. ubiquitin ACTIVATING enzyme (E1)

2. ubiquitin-CONJUGATING enzyme (E2)

3. ubiquitin- protein LIGASE (E3)

SEQ Ubiquitination

1. Using ATP, the ubiquitin- ________ enzyme (E1) adenylates ubiquitin and transfers a _______ group of a ____ residue to E1

2. Ubiquitin- __________ enzyme (E2) transfers ubiquitin to one of its own _________ groups

3. 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

1. activating sulfhydryl cys

2. conjugating sulfhydryl

3. ligase E

4. 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

1. E3 remains bound allowing for a chain of ubiquitin to form

2. 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?

1. n-degron

2. cyclin destruction boxes

3. 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?

1. amino acids —> proteins

2. amino acid—> remove amino group —> urea —> carbon skeleton used for CARBOHYDRATES

3. amino acid —> remove amino group—> carbon skeleton used for FATS

4. 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?

1. NH4+ + HCO3- → carbomyl phosphate

2. carbomyl phosphate + ornithine —> citrulline

3. citrulline + aspartate —> arginine succinate

4. arginine succinate—> arginine + fumarate

5. arginine —> UREA +

first two steps in mitochondria

rest in CYTOPLASM starting from aspartate + citrulline —> arginine succinate

UREA CYCLE: which enzymes catalyze each step?

1. NH4+ + HCO3- ———→ carbomyl phosphate

2. carbomyl phosphate + ornithine ——> citrulline

3. citruline + aspartarte —> arginino succinate

4. arginino succinate —> Arginine + Fumarate

5. Arginine —> UREA + ornithine

1. carbomyl phosphate synthetase I

   N-acytelglutamate

   N-acytelglutamate synthetase (if high amino acid)

2. orthinine transcarbomylase

3. arginosuccinate synthetase + ATP

4. argininosuccinase (cleaves)

5. 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

1. gluconeogenesis form glucose from oxaloacetate

2. 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