Urea Cycle and It's Regulation

 Definition

• Ammonia is a toxic product of nitrogen metabolism which should be removed from our body. The urea cycle orrnithine cycle converts excess ammonia into urea in the mitochondria of liver cells. The urea forms, then enters the blood stream, is filtered by the kidneys and is ultimately excreted in the urine.
• The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions that produces urea (NH2)2CO from ammonia (NH3). This cycle occurs in ureotelic organisms. The urea cycle converts highly toxic ammonia to urea for excretion.
• This cycle was the first metabolic cycle to be discovered by Hans Krebs and Kurt Henseleit, in 1932 five years before the discovery of the TCA cycle. 
• This cycle was described in more detail later on by Ratner and Cohen. 

Function

• Amino acid catabolism results in waste ammonia. 
• All animals need a way to excrete this product. 
• Most aquatic organisms, or ammonotelic organisms, excrete ammonia without converting it.
• Organisms that cannot easily and safely remove nitrogen as ammonia convert it to a less toxic substance, such as urea, via the urea cycle.
•  Urea produced by the liver is then released into the bloodstream, where it travels to the kidneys and is ultimately excreted in urine.
• The urea cycle is essential to these organisms, because if the nitrogen or ammonia are not eliminated from the organism it can be very detrimental.
•  In species including birds and most insects, the ammonia is converted into uric acid or its urate salt, which is excreted in solid form.

Reactions 

• The entire process converts two amino groups, one from NH+4

 and one from Aspartate, and a carbon atom from HCO−

3, to the relatively nontoxic excretion product urea.

• This occurs at the cost of four "high-energy" phosphate bonds (3 ATP hydrolyzed to 2 ADP and one AMP). 
• The conversion from ammonia to urea happens in five main steps. 
• The first is needed for ammonia to enter the cycle and the following four are all a part of the cycle itself. 
• To enter the cycle, ammonia is converted to carbamoyl phosphate. 
• The urea cycle consists of four enzymatic reactions: one mitochondrial and three cytosolic.This uses 6 enzymes.

Reactions of the Urea Cycle

1 L-ornithine

2 carbamoyl phosphate

3 L-citrulline

4 argininosuccinate

5 fumarate

6 L-arginine

7 urea

L-Asp L-aspartate

CPS-1 carbamoyl phosphate synthetase I

OTC Ornithine transcarbamoylase

ASS argininosuccinate synthetase

ASL argininosuccinate lyase

ARG1 arginase 1

First reaction: entering the urea cycle 

Before the urea cycle begins ammonia is converted to carbamoyl phosphate. The reaction is catalyzed by carbamoyl phosphate synthetase I and requires the use of two ATP molecules.[1] The carbamoyl phosphate then enters the urea cycle.

Steps of the urea cycle 

• Carbamoyl phosphate is converted to citrulline. With catalysis by ornithine transcarbamoylase, the carbamoyl phosphate group is donated to ornithine and releases a phosphate group.
• A condensation reaction occurs between the amino group of aspartate and the carbonyl group of citrulline to form argininosuccinate. This reaction is ATP dependent and is catalyzed by argininosuccinate synthetase.
• Argininosuccinate undergoes cleavage by argininosuccinase to form arginine and fumarate.
• Arginine is cleaved by arginase to form urea and ornithine. The ornithine is then transported back to the mitochondria to begin the urea cycle again.
• 
  

Overall reaction equation 

In the first reaction, NH+

4 + HCO3- is equivalent to NH3 + CO2 + H2O.

Thus, the overall equation of the urea cycle is:

NH3 + CO2 + aspartate + 3 ATP + 3 H2O → urea + fumarate + 2 ADP + 2 Pi + AMP + PPi + H2O

Since fumarate is obtained by removing NH3 from aspartate (by means of reactions 3 and 4), and PPi + H2O → 2 Pi, the equation can be simplified as follows:

2 NH3 + CO2 + 3 ATP + 3 H2O → urea + 2 ADP + 4 Pi + AMP

Note that reactions related to the urea cycle also cause the production of 2 NADH, so the overall reaction releases slightly more energy than it consumes. The NADH is produced in two ways:

• One NADH molecule is produced by the enzyme glutamate dehydrogenase in the conversion of glutamate to ammonium and α-ketoglutarate. Glutamate is the non-toxic carrier of amine groups. This provides the ammonium ion used in the initial synthesis of carbamoyl phosphate.
• The fumarate released in the cytosol is hydrated to malate by cytosolic fumarase. 
• This malate is then oxidized to oxaloacetate by cytosolic malate dehydrogenase, generating a reduced NADH in the cytosol. Oxaloacetate is one of the keto acids preferred by transaminases, and so will be recycled to aspartate, maintaining the flow of nitrogen into the urea cycle.

We can summarize this by combining the reactions:

CO2 + glutamate + aspartate + 3 ATP + 2 NAD++ 3 H2O → urea + α-ketoglutarate + oxaloacetate + 2 ADP + 2 Pi + AMP + PPi + 2 NADH

• The two NADH produced can provide energy for the formation of 5 ATP , a net production of two high-energy phosphate bond for the urea cycle. However, if gluconeogenesis is underway in the cytosol, the latter reducing equivalent is used to drive the reversal of the GAPDH step instead of generating ATP.
• The fate of oxaloacetate is either to produce aspartate via transamination or to be converted to phosphoenolpyruvate, which is a substrate for gluconeogenesis.

Products of the urea cycle 

• As stated above many vertebrates use the urea cycle to create urea out of ammonium so that the ammonium does not damage the body. Though this is helpful, there are other effects of the urea cycle. 
• For example: consumption of two ATP, production of urea, generation of H+, the combining of HCO3- and NH4+ to forms where it can be regenerated, and finally the consumption of NH4+.

Regulation

The biosynthesis of urea is regulated mainly by two factors, the amounts of urea cycle enzymes and the concentrations of acetyl-glutamate and ornithine

N-Acetylglutamic Acid 

• The synthesis of carbamoyl phosphate and the urea cycle are dependent on the presence of N-acetylglutamic acid (NAcGlu), which allosterically activates CPS1.
•  NAcGlu is an obligate activator of carbamoyl phosphate synthetase.
• Synthesis of NAcGlu by N-acetylglutamate synthase is stimulated by both Arg, allosteric stimulator of NAGS, and Glu, a product in the transamination reactions and one of NAGS's substrates, both of which are elevated when free amino acids are elevated. 
• So Glu not only is a substrate for NAGS but also serves as an activator for the urea cycle.

Substrate Concentrations

• The remaining enzymes of the cycle are controlled by the concentrations of their substrates. Thus, inherited deficiencies in cycle enzymes other than ARG1 do not result in significant decreases in urea production (if any cycle enzyme is entirely missing, death occurs shortly after birth). Rather, the deficient enzyme's substrate builds up, increasing the rate of the deficient reaction to normal.
• The anomalous substrate buildup is not without cost, however. The substrate concentrations become elevated all the way back up the cycle to NH+4, resulting in hyperammonemia.

Schimke1 pointed out that the contents of all the urea cycle enzymes in the liver were directly proportional to the daily consumption of protein, then the activities of urea cycle enzymes are an important regulatory factor of the urea cycle. On the other hand, other investigators2–4 reported that the concentration of acetylglutamate, an allosteric activator of carbamylphosphate synthesis, and of ornithine, the rate limiting intermediate, changed under various dietary conditions and suggested that these amino acids play a role in the regulation of urea synthesis. 

We reported that ornithine and acetylglutamate play a more important role in the regulation of urea synthesis especially shortly after the dietary change. In the liver of rats subjected to acute dietary transitions from high to low protein or vice versa, the concentrations of ornithine and acetylglutamate changed greater and prior to the activity changes of urea cycle enzymes. The rate of urea synthesis from ammonium salt as a substrate were greatly changed in the perfused liver and correlated with the changes in the concentration of ornithine in the liver after the dietary changes5.

 Arginine derived from dietary protein is thought to be the main source of ornithine and also the cause of changes in acetylglutamate3–5. However, other factors must be involved in the regulation of the concentration of ornithine, since the concentration of orni-thine as well as acetylglutamate increased after the intraperitoneal injection of the ammonium salt without altering either arginine or protein input

Images