M4: Urea Cycle L26

Question 1

Which of the following is (are) involved in net deamination reactions?
A. Glutamine synthetase
B. Glutaminase
C. Aspartate transaminase
D. Alanine transaminase
E. Glutamate dehydrogenase

Answer 1

B. Glutaminase

E. Glutamate dehydrogenase

Question 2

What prosthetic group(s) serve(s) as co-factor(s) for transaminases?
A. PLP
B. PMP 
C. PNP 
D. POP 
E. PPP
F. VitB6 
G. Vit B12

Answer 2

A. PLP

B. PMP

Question 3

Which of the following prefer(s) a-KG as a substrate?
A. Glutaminase
B. Glutamate dehydrogenase
C. ALT2
D. Aspartate transaminase
E. Tyrosine aminotransferase
F. Proline transaminase

Answer 3

D. Aspartate transaminase

E. Tyrosine aminotransferase

Question 4

Where does the urea cycle take place? What’s its purpose?

Answer 4

Happens in the liver. Generates Urea to detoxify NH3 so that it does not become toxic to the brain.

Question 5

Describe what happens in the mitochondrial matrix in the liver before the urea cycle.

Answer 5

1. Glutamine from the liver gets into the mitochondria and the glutaminase reaction makes NH4 and glutamate from glutamine. (Not transamination)
2. Deamination of Glutamate via Glutamate dehydrogenase (GDH) to release alpha-KG and another NH4+.
3. The 1st substrate of the urea cycle, Carbamoyl phosphate is formed via Carbamoyl Phosphate Synthetase 1 (CPS1), HCO3- (CO2), 2ATPs, and the previously generated NH4+.

Question 6

Describe the CPS1 reaction.

Answer 6

Carbamoyl phosphate synthetase 1 (CPS1):
Irreversible => CPS1 is the rate-limiting step
1. Bicarbonate & an ATP (input of energy), to prime the bicarbonate carbon with a phosphate so that it can bind the NH3 group
2. Inorganic phosphate released when ammonium binds. Carbamate intermediate is made
3. Use a second ATP to prime carbamate with a phosphate. ADP is released.
4. Carbamoyl phosphate is generated (high energy intermediate bc it has been primed).

Question 7

How is CPS1 regulated? Slide 24, L26

Answer 7

Irreversible => CPS1 is the rate-limiting step
• Occurs at high ATP
• Regulated (activated) by the allosteric activator NAG (N-acetylglutamate)
• NAG is produced by NAGS (NAG Synthase)
• NAGS is stimulated by Arginine (amino acid)
=> NAG production is proportional to [a.a.] in circulation

To activate CPS1: you need NAG, you also need a functional NAGS to produce NAG.
NAGS: stimulated by arginine (amino acid). Glutamate + acetyl-CoA + NAGS => NAG

So if you have glutamate, acetyl CoA, and arginine all in abundance in your cell, you have 2 abundant amino acids, it is probably a sign that you need to detoxify the NH3 being released from amino acids. This is why you need to activate the CPS1 (to activate urea cycle).

Question 8

How is NAG synthesized? Where?

Answer 8

Glutamate + acetyl-CoA => NAG
Via NAG synthase (NAGS) stimulated by arginine.
In the mitochondrial matrix in the liver.

Question 9

Why is the urea cycle compartmentalized?

L26, Slide 25.

Answer 9

There are 2 similar but different enzymes:

Carbamoyl Phosphate Synthetase 1 (CPS-1):
• UREA synthesis
• Mitochondrial
• Absolutely needs NAG
• Uses NH4+/NH3 directly

Carbamoyl Phosphate Synthetase 2 (CPS-2):
• Pyrimidine synthesis (to make nucleotides)
• Cytoplasmic
• No need for NAG
• Uses Glutamine as NH3 donor (doesn’t use NH3 directly)

Compartmentalization allows for Nucleotides and urea to be synthesized at the same time. No interference between the two pathways. If they weren’t compartmentalized, there would be a problem because glutamine is a substrate for both, and they would compete. Having them separate means they are not competing and they can both happen.

Question 10

What abnormalities can cause hyperammonia in humans?

Answer 10

Abnormalities associated with the urea cycle.

1. CPS-1 deficiency (missense mutation): HyperAmmonemia, liver failure and death at a very young age in those that are homozygous for the mutation (e.g. 4 days after birth). If you have a point mutation and are heterozygous for the CPS1 deficiency, then you can probably survive, but if you decide to intake a lot of amino acids (ex: if ur a body builder) then you will run into problems because there will be a delayed clearance of excess ammonia in your blood
2. NAGS deficiency (nonsense mutation): Closely resembles CPS-1 deficiency. Since you cant synthesize NAG very well, there will be delayed or poor activation of CPS1.
3. Glutamate dehydrogenase (GDH) gain-of-function mutation in the GTP allosteric binding site:
   GDH can’t be shut down =>HyperAmmonia/HyperInsulinism because more NH4+ will be produced which will clog CPS1. (problem with the maintenance of NH3 homeostasis).

Question 11

Study the Urea cycle from the ppt.

Answer 11

Slide 30-34. Lecture 26.

Question 12

The urea cycle uses aspartate as a substrate to donate an amino group. Where does the aspartate come from?

Answer 12

Glutamate (a.a. 1) + oxaloacetate (K.A. 2) => a-KG (K.A. 1) + Aspartate (a.a. 2) via aspartate aminotransferase in the mitochondria.

Legend:
a.a. = amino acid
K.A. = Keto acid

The aspartate formed is exported out of the mitochondrial matrix and into the cytosol to be used as a substrate for the cytosolic portion of the urea cycle.

Question 13

Where do the nitrogens in urea come from? (2 nitrogens)

Answer 13

1) From oxidative deamination of Glutamate.
2) From Aspartate.
However, both groups ultimately come from Glutamate!

The aspartate comes from the transamination reaction in the mitochondria using glutamate. the aspartate formed is then used as a nitrogen donor in the urea cycle later on.

Question 14

What is the link between the urea cycle and the CAC?

Answer 14

1. the oxaloacetate used to make aspartate comes form the CAC.
2. The fumarate released from the urea cycle can enter the CAC directly as fumarate or is converted to malate which also enters the CAC.

Question 15

What is the energy cost of the urea cycle?

Answer 15

Synthesis of 1 urea molecule requires hydrolysis of 4 high-energy phosphate groups (2ATP and 1 PP1 from CPS1 step and 1 ATP from citrulline to arginosuccinate).

Overall the rxn is not too bad because the urea cycle consumes 4ATP, but the GDH reaction makes an NADH which can make 2.5 ATP. Also, Fumarate made in the urea cycle can feed into he CAC to generate another NADH (another 2.5 ATP). So 2.5 + 2.5 -4= +1ATP generated. So the energy cost and energy production is pretty much balanced.