Protein Metabolism: N Disposal - Madura 3/10/16 Flashcards
protein degradation
- two types
- why?
two diff locations: different, but both highly controlled/reg’d with many similarities
- extracellular (in gut)
- intracellular (in tissues)
- compartmentalized
- catalyzed by proteases (formerly zymogens)
why?
- intracellular: wear&tear, don’t need them anymore
- extracellular: digestion of dietary proteins so you can replace old proteins and rebuild them
basics of extracellular protein digestion
basics of protein catabolism
dietary proteins broken down into smaller units that can cross int epithelial cell membrane → free a.a.s transported to liver for processing
- degradation of proteins → amino acids
- alpha amino group removed
- ammonia eliminated (as urea)
- carbon chain recycled into carb/fat metabolism (energy production or storage)
similarities and diffs of carb/fat metabolism and protein metabolism
storage
- carbs/fats can be stored
- proteins cannot be stored
all can be tapped for energy, but…
- protein, a.a.s, and ammonia cant be stored → excess has to be metabolized so N group is removed and excreted as urea
- some N can be assimilated into other metabolic pdts (minor fraction)
- ammonia is toxic, is rapidly excreted
- toxicity resembles alcohol tox
A1 equilibrium in protein uptake/excretion:
amino acid pool
steady state amount of free, available a.a.s for the synthesis of new proteins and other building blocks
- net of approx 100g in any given individual
where is it coming from?
- body breaks down approx 400g of protein per day → rebuilds most of it
- addt’l 100g coming in through diet
excess? has to be excreted through forming urea
A1 equilibrium in protein uptake/excretion:
N balance
balance between nitrogen intake in diet and excretion in urine
negative N balance: excretion > intake
- sepsis
- muscle wasting
- fasting
- deficiency of essential a.a.s (HILK MV FTW)
positive N balance: intake > excretion
- pregnancy
- growth
- tumor
A2 degradation of dietary proteins
three sites of protease synthesis
primary site of protease action
- stomach (pepsin)
- food has been chewed, proteins starting to come apart
- acidic pH of the stomach helps them become denatured
- pancreas
* signals received while proteins are in stomach result in release of zymogens into sm int - small intestine epi cells
most of the action occurs in sm intestine, where all proteases are active (optimal pH)
- if they were autocatalyzed and active before this point, you’d see death of cells (ex. pancreas)
A2 degradation of dietary proteins
key enzymes and activation
stomach: pepsinogen → pepsin
pancreas: zymogens activated by enteropeptidases in small intestine → remove Pro sequences that block catalytic site
- chymotrypsinogen → chymotrypsin (hydrophobic)
- trypsinogen → trypsin (basic)
- proelastase → elastase (aliphatic)
major player: trypsin
- activated by enteropeptidase
- also autocatalytic (can activate itself…once trypsin is activated in the first place)
- once active, trypsin can…
- start degrading dietary proteins
- start activating other zymogens
A2 degradation of dietary proteins
protease action
why is the intestine spared?
- act in extracellular site (gut lumen)
nonspecific protease fx : prefer to break certain peptide bonds, but don’t discriminate based on whole protein identity!
- food gets to stomach, digestion starts, CCK triggers pancreatic protease release → RAPID ACTIVATION AND WORK OF PROTEASES → generate some free a.a.s, dipeptides, tripeptides that can be absorbed into int epi cells
why is intestine spared from the indiscriminate destructive action of zymogens??? MUCUS!
A2 degradation of dietary proteins
absorption and transport of products for a.a. degradation
potential problems
at this point, have a collection of a.a.s and small peptides that are moved into int epi cells by transporters
- only free a.a.s can be moved into circulation, so dipeptidases, tripeptidases inside epi cells go to work
- a.a.s enter portal vein → transport to liver
*issues with transporters can lead to reduced uptake of specific a.a.s
*high urea in circulation can cross into intestine, be broken down by urease → release ammonia
- rapid diffusion of ammonia into circ → CNS toxicity
do all sources of proteins have equivalent nutritional value?
no
plants have low quantity of certain essential a.a.s
degradation of proteins intracellularly requires ATP, but degradation of proteins extracellularly (gut) does not
why?
proteins can’t be degraded until they are unfolding, unfolding requires ATP (intracellularly)
in gut, acidic environment take care of the unfolding, so you don’t have to spend ATP on it!
A3 removal of alpha amino group
transamination reaction
- why important?
- location
- enzyme [required cofactor]
- reactants, products
removing alpha amino group means you’re freeing ammonia, which is v toxic
- need to have a system in place to capture and control ammonia you release
a. a. + ketoacid acceptor → alpha-ketoacid + Glu/Ala/Asp
transamination reaction :
(family of 7-8) aminotransferases remove alpha-amino group from a.a. and transfer it to a ketoacid acceptor → alpha-ketoacid + a.a.
- occurs in liver
- reversible reaction, direction dictated by energy needs
- essential cofactor: pyridoxal phosphate (B6)
- generates 20 alpha-ketoacids, but all are shuttled into one common pathway!
*alpha-KG is the common acceptor for the majority of these rxns, glutamate is a common final carrier of the NH3 you removed from the a.a.
- can also be pyruvate → Ala; OAA → Asp
A3 removal of alpha amino group
transamination reaction
- role of pyridoxal phosphate
essential cofactor for aminotransferase to do its thing
- is the middleman for NH3 transfer
- picks NH3 up from a.a.
- hands it off to acceptor ketoacid (typically alpha-KG)
- no energy required
(can also form a transient Schiff base with the enzyme)
**have not altered N pool - just mobilized the NH3**
A3 removal of alpha amino group
3 key a.a./ketoacid pairs
- major roles
1. alpha-ketoglutarate ⇔ glutamate
- key in transamination rxn
2. pyruvate ⇔ alanine
- key in glucose-alanine cycle: capture of ammonia in peripheral tissue and safe transport to liver
3. oxaloacetate ⇔ aspartate
- key in urea cycle → donates a second N into cycle
*all three acceptor ketoacids are TCA cycle intermediates!
- link: when energy is v low, body needs to start breaking down muscle protein, which is where they come in
A3 removal of alpha amino group
role of glutamate (and alpha-KG)
most alpha-NH3 groups are transferred to alpha-KG → glutamate
- alphaKG is a TCA intermediate: link to energy utilization/need
- temp reservoir, if needed: Glu is able to pick up another NH3 to hold temp (Glu → Gln)
- precursor for N-acetylGlu: allosteric activator of urea cycle
- N gets assimilated into urea via oxidative deamination, followed by transamination : OAA → Asp
A3 removal of alpha amino group
getting rid of NH3, making urea
only organ that can make urea : LIVER
- only organ that should have any appreciable level of ammonia, bc its the only organ that’s equipped w/ enzymes to process it
- has oxidative and non-ox pathways to release NH3
Glu → alpha-KG + NH3 [glutamate DH; need NAD+]
A3 removal of alpha amino group
glucose-alanine cycle
in active muscle tissue, lots of pyruvate
- used as a tool to sequester NH3 (also high) and send it to liver for conversion to urea and excretion
in sk muscle: convert pyruvate → Ala and transport it in circulation to liver
in liver: convert Ala → pyruvate [by transferring it through alpha-KG → Glu]
- pyruvate can be converted into glucose, which can be sent back to muscle tissue for use as egy → pyruvate made again
A4 synthesis of urea
what’s the point?
compartmentalized model
- characteristics of rxns
- key enzyme & location
point: take toxic ammonia and turn it into a benign pdt for excretion
rxn separated in 2 compartments
process starts in mito matrix (addition of first N group), proceeds into cytosol (add second N group)
- unidirectional (transporters send it out of mito into cytosol; won’t send back in)
- each addition of N group requires ATP - irreversible
cytosolic rxns of urea cycle run, eventually…get arginine, which is processed by arginase to produce urea → on to kidneys for excretion
- NH3 comes from Asp (via Glu)
- intermediates that are also ints in TCA cycle
- arginase is only expressed in liver (!), which is tasked with production of urea
A4 synthesis of urea
regulation
only want urea synthesis to occur when there’s an excess of a.a.s that needs to be broken down
- urea synthesis rises in response to increase of Arg in a.a. pool (indicates protein consumption → excess that needs to be gotten rid of)
Arg stimulates enzyme that takes glutamate → N-acetylglutamate, cofactor of CPS1 (rate limiting factor of whole urea cycle!)
- CPS1 converts ornithine → citrulline, transportable into cytosol, where rest of cycle can take place
A4 synthesis of urea
process summary
summary:
- digestion of protein → rise in Arg → stimulation of Glu → N-acetylglutamate
- N-acetylglutamate is a cofactor for CPS1, which is the rate-limiting step
- CPS1 acts in mito matrix, adds N group to convert ornithine → citrulline, transportable form that can get from mito matrix into cytoplasm!
- cytosolic rxns eventually produce arginine, which arginase converts → urea (excreted) + ornithine (moved back into mito matrix for repeat)
A4 synthesis of urea
key features
- irreversible
- energy-requiring
- compartmentalized (mito matrix, unidirectional transit into cytosol)
- key enzymes only found in liver
- rate limiting step: CPS1 [ornithine → citrulline] - adds first N group and gets molecule into form that can be transported out into cytosol
- arginase [Arg → urea + ornithine] - releases urea
- positively regulated : Arg positively regulates production of cofactor for rate-limiting step (N-acetylglutamate)
- Glutamate is source of both N
- direct: oxidative deamination of ammonia
- indirect: aminotransferase moves it from Glu → Asp, which transfers it into the cycle in the cytosolic rxns
- defects can cause hyperammonemia
defects in urea cycle
cause hyperammonemia
mutation in CPS1
mutation in n-acetylglutamate synthase
- see reduced flux through pathway, especially in damaged liver
symptoms will be similar to alc intox, but for diff reasons!
- slurred speech, altered gait, loss of balance, severe: coma
what if urea system gets backed up → ammonia levels rise?
in healthy individ, not a problem due to built-in overcapacity of enzyme
in severe cases or people with compromised liver fx: ammonia toxicity
spike in ammonia leads cells to…
- transport it to liver in circulation (ex. like in alanine-glucose cycle)
- cells in any tissue can turn Glu → Gln and/or Asp → Asn (throw an extra NH3 group on there temporarily)
- requires energy, enzymes :
- in liver: rxn can be reversed by glutaminase, asparaginase
what if kidney fails and can’t excrete urea → buildup of urea in blood
what can happen?
urea can get to intestine, get past int epithelium into lumen
bacterial enzyme, urease can hydrolyze urea → NH3
- NH3 can get into circulation and cause NH3 toxicity!
in a healthy individual, would try to control this spike in blood NH3 by over-loading Glu → Gln, Asp → Asn and getting it to the liver
