Protein Metabolism: N Disposal - Madura 3/10/16

Question 1

protein degradation

• two types
• why?

Answer 1

two diff locations: different, but both highly controlled/reg’d with many similarities

1. extracellular (in gut)
2. 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

Question 2

basics of extracellular protein digestion

basics of protein catabolism

Answer 2

dietary proteins broken down into smaller units that can cross int epithelial cell membrane → free a.a.s transported to liver for processing

1. degradation of proteins → amino acids
2. alpha amino group removed
3. ammonia eliminated (as urea)
4. carbon chain recycled into carb/fat metabolism (energy production or storage)

Question 3

similarities and diffs of carb/fat metabolism and protein metabolism

Answer 3

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

Question 4

A1 equilibrium in protein uptake/excretion:

amino acid pool

Answer 4

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

Question 5

A1 equilibrium in protein uptake/excretion:

N balance

Answer 5

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

Question 6

A2 degradation of dietary proteins

three sites of protease synthesis

primary site of protease action

Answer 6

1. stomach (pepsin)

• food has been chewed, proteins starting to come apart
• acidic pH of the stomach helps them become denatured

2. pancreas
   * signals received while proteins are in stomach result in release of zymogens into sm int
3. 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)

Question 7

A2 degradation of dietary proteins

key enzymes and activation

Answer 7

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

Question 8

A2 degradation of dietary proteins

protease action

why is the intestine spared?

Answer 8

• 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!

Question 9

A2 degradation of dietary proteins

absorption and transport of products for a.a. degradation

potential problems

Answer 9

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

Question 10

do all sources of proteins have equivalent nutritional value?

Answer 10

no

plants have low quantity of certain essential a.a.s

Question 11

degradation of proteins intracellularly requires ATP, but degradation of proteins extracellularly (gut) does not

why?

Answer 11

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!

Question 12

A3 removal of alpha amino group

transamination reaction

• why important?
• location
• enzyme [required cofactor]
• reactants, products

Answer 12

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

Question 13

A3 removal of alpha amino group

transamination reaction

• role of pyridoxal phosphate

Answer 13

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**

Question 14

A3 removal of alpha amino group

3 key a.a./ketoacid pairs

• major roles

Answer 14

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

Question 15

A3 removal of alpha amino group

role of glutamate (and alpha-KG)

Answer 15

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

Question 16

A3 removal of alpha amino group

getting rid of NH3, making urea

Answer 16

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+]

Question 17

A3 removal of alpha amino group

glucose-alanine cycle

Answer 17

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

Question 18

A4 synthesis of urea

what’s the point?

compartmentalized model

• characteristics of rxns
• key enzyme & location

Answer 18

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

Question 19

A4 synthesis of urea

regulation

Answer 19

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

Question 20

A4 synthesis of urea

process summary

Answer 20

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)

Question 21

A4 synthesis of urea

key features

Answer 21

1. irreversible

• energy-requiring
• compartmentalized (mito matrix, unidirectional transit into cytosol)

2. 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

3. positively regulated : Arg positively regulates production of cofactor for rate-limiting step (N-acetylglutamate)
4. 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

5. defects can cause hyperammonemia

Question 22

defects in urea cycle

Answer 22

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

Question 23

what if urea system gets backed up → ammonia levels rise?

Answer 23

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

Question 24

what if kidney fails and can’t excrete urea → buildup of urea in blood

Answer 24

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