Lecture 14: Amino Acid Deamination

Recall: Protein store in a 70 kg human is 6 kg (fat = 15 kg, glycogen/glucose < 1g)
- Energy content = 17 kJ/g (same as carbs)
- Total kJ = 100 000 

With a few exceptions, the first step in amino acid degradation involves the removal of alpha-amino group to give the corresponding alpha-keto acid:

Transaminase vs amidotransferase
§transaminase is an enzyme that catalyzes a reaction between a keto acid and an amino acid.
- An amino group and an =O are exchanged in this rxn
§ amidotransferase is an enzyme that catalyzes the removal of an amino group from a donor molecule and transfer it to a substrate.
- Take amino from one substrate and transfer it to another substrate

All these reactions are reversible
What we're doing here is...
1) Amino acids become ammonia
2) the ammonia is transferred to the liver from the muscles and other tissues to be excreted in the urea cycle

1) Transamination
Transaminases are bound to the PLP coenzyme

Step "0" is to form the PLP-transaminase complex from PLP + Transaminase

Step 1: amino group is carried onto the transaminase by latching onto the coenzyme PLP
- the PLP complex becomes Pyridoxamine phosphate (PMP)

Step 2: Transfer amino group from PLP (actually PMP at this moment) onto a different alpha-keto acid: alpha-ketoglutarate
- Done by a transaminase
- Result = glutamate

1) PLP-dependent transamination reaction is reversible
2) Any amino acids can be used as substrate for the first stage (forward) reaction
3) alpha-ketoglutarate is the major alpha-keto acid substrate in the second stage (reverse) reaction

2) Oxidative Deamination
Glutamate will give the ammonia to other molecules of the urea cycle
Glutamate dehydrogenase (GDH) catalyzes this deamination (NOT transamination)
- Reversible reaction 
- Generates 1 NADPH or 1 NADH (depending on where the reaction occurs)

3) Transport of Ammonia to Liver
Urea cycle appens in the Liver (converts ammonia -> urea); all organs degrade protein to form ammonia; accumulation is toxic
- To transport to liver = glutamine synthetase (b/c glutamine is non-toxic compound until in liver)

Most tissues use glutamine synthetase (add an amino group to glutamate to form glutamine
- Enzyme = Glutamine synthetase
- Use 1 molecule of ATP
Glutamine will travel through blood; converted back to glutamate via glutaminase

Muscles use same system as the Cori Cycle
- Lots of pyruvate around in muscle; can't be converted back to glucose (recall gluconeogenesis only in liver)
- Converts pyruvate to alanine
- Transamination converts pyruvate into alanine (which is also non-toxic)
-- Catalyzed by alanine transaminase
-- The keto in this reaction is the pyruvate (??? I think it's actually just the alpha-ketoglutarate)
- In the liver, the reverse reaction occurs to make alanine -> pyruvate, the ammonia goes onto glutamate
- Glucose-Alanine cycle relies on pyruvate (while Cori relies on lactate)

Recall Cori Cycle: Glucose -> 2 pyruvate -> 2 lactate, travels in blood to liver, where it's converted back to glucose; the glucose goes through bloodstream back to muscles
- In glucose-alanine cycle, pyruvate becomes alanine instead of lactate and in the process converts glutamate to alpha-ketoglutarate (and back in the liver)

CORI CYCLE: Utilization of lactate produced by anaerobic glycolysis in the muscles
GLUCOSE-ALANINE CYCLE: Utilization of amino acid metabolites released from muscles

So, this Glucose-Alanine Cycle provides two jobs in muscles:
1) carry ammonia to the urea cycle
2) Replenish stock of glucose by carrying pyruvate -> alanine to liver and letting gluconeogenesis happen in there

4) Urea Cycle!
Also called the Krebs-Henseleit Urea Cycle; IN THE LIVER
- Discovered 5 years before the TCA cycle; they use common components
Cycle starts and ends with ornithine, which carries molecules up until a point where it releases urea, recreating ornithine again

Five reactions: the first two happen in the mitochondria
- It seems like ammonia gets into the mitochondria by a simple diffusion through the membrane
STEP 1: Bicarbonate, attach 1 molecule of ammonia, end product = carbamoyl phosphate
Catalyzed by CPS1
Uses 2 ATP
Phosphorylation of bicarbonate, then a release of organic phosphate to produce carbamate, then produce carbamoyl phosphate
RATE-LIMITING STEP is what is entering  the cycle
STEP 2: The carbamoyl phosphate attaches to amino group on ornithine; releases organic phosphate
- Produces citrulline
- Catalyzed by OTC (ornithine transcarbamylase)

STEP 2b: Ornithine/citrulline Transport
- Done by ORC1 (Ornithine carrier 1) which transports ornithine, lysine, arginine, and citrulline
- Transporter between mitochondria and cytosol
STEP 3: Citrulline + aspartate (from a mitochondrion) = argininosuccinate
- Catalyzed by argininosuccinate synthetase
- Uses equivalent of 2 ATP (ATP -> AMP + PP is TWO phosphates); releases pyrophosphate

STEP 4: argininosuccinate is cut into fumarate and arginine
- done by argininosuccinase

STEP 5: cleave urea off of the arginine and also reproduces ornithine
- Uses 1 molecule of H2O
- Catalyzed by arginase

Regulating Pathway Flux?
Nitrogen flux is based on diet (recall that amino acids CANNOT be stored)
We break them down when we eat them, we break them down get rid of waste as urea

Regulator = N-acetylglutamate (allosterically activates CPS1 which forms carbamoyl phosphate)
- This molecule is made from glutamate + Acetyl-CoA
- Remember, glutamate can be a carrier of ammonia; Lots of glutamate = lots of ammonia, so the N-acetylglutamate will be produced if there's lots of glutamate.

SECOND FUNCTION: through 4 steps, N-acetylglutamate can be converted to ornithine; so it's serving as a store of ornithine as well

Urea Cycle Summary
1) Urea cycle is confined to liver
2) Reactions occur in both the mitochondria (1-2) and the cytosol (3-5)
3) Ornithine transport from cytosol into mitochondrion is carried out by a translocase (transporter)
4) Amino groups of urea are derived from NH3 and aspartate, and the carbon atom is derived from CO2
5) Carbon skeleton product can be used for gluconeogenesis

Thinking question...
Diet of all amino acids except arginine -> develops ammonia toxicity
a) Role of fasting = lower blood glucose levels to induce them to rely on their amino acids
b) What caused ammonia level to rise? Oxidative deamination of amino acids produces ammonia at the end
c) Why was lack of arginine a cause for ammonia toxicity? Since arginine levels are low, and arginine is an intermediate of the urea cycle, the urea cycle slowed as well
- Arginine is an essential amino acid; cats synthesize ornithine directly from arginine, so cycle is blocked in absence of arginine
d) Why can ornithine be substituted for arginine? It is also an intermediate of the urea cycle so we will generate more arginine

The Krebs Bicycle
Both the Urea and Krebs Cycle are interconnected
- The aspartate for step 3 in the urea cycle comes from the Krebs cycle
- The fumarate from step 4 of the urea cycle is converted into malate, which goes back into the Krebs cycle

Amino Acid Breakdown - What do we do with the carbon skeleton after it is deaminated?
Most amino acids can be degraded into one of seven metabolic intermediates:
1) Oxaloacetate
2) alpha-ketoglutarate
3) Succinate
4) Fumarate
5) Pyruvate
6) Acetyl-CoA
7) Acetoacetate

Some amino acids are gluconeogenic: can be degraded to intermediates 1-5 (so they're glucose precursors)
- Any of them can be converted to oxaloacetate which can become glucose
- E.g. Alanine, serine, cysteine, threonine become pyruvate
Others are ketogenic: can be degraded to acetyl-CoA or acetoacetate so then they can be converted to ketones or FAs
All AA carbon skeletons can be used as a source of energy.
- Example of ketogenics: Leucine and lysine become acetyl-CoA through HMG-CoA 

§ The amino groups and the carbon skeletons are catabolized via different pathways
§ The urea cycle and TCA cycle are interconnected
§ AA catabolism can replenish the TCA cycle intermediates (recall anaplerosis = act of replenishing TCA cycle intermediates that have been extracted for biosynthesis)
§ Amino acid degradation represents a minor (10-15% of total) yet important pathway for energy production