Urea Cycle: Steps, End Products, and Functions

Amino nitrogen, a key component in the synthesis of amino acids or new nitrogenous products, can be toxic to the human body if not utilized to create new compounds. To prevent this, ureotelic organisms like humans excrete amino nitrogen as urea. This process, known as the urea or ornithine cycle, involves channeling the amino groups or ammonia into the formation of urea. The mitochondria and cytosol of hepatocytes (liver cells) are where the urea cycle occurs, serving as a crucial mechanism for maintaining the body’s nitrogen balance. 

Hans Krebs and medical student associate Kurt Henseleit discovered this pathway in 1932. The urea produced in the liver passes into the bloodstream and thus to the kidneys and excreted into the urine. 

Urea production occurs in five enzymatic steps and begins in the mitochondria of hepatocytes. However, three of the steps of the urea cycle, including the final step, occur in the cytosol. Fumarate and urea are some of the end products of the urea cycle. 

There is interlinking between the citric or Krebs cycle and the urea cycle due to the byproduct fumarate produced during the cycle. Likewise, several enzymes of the citric cycle are present as isozymes in the urea cycle. 

Two levels of regulation occur in the urea cycle: one is the dietary intake of proteins, and the other is allosteric regulation at one of the key enzymes. 

Enzymes Involved In Urea Cycle

The urea cycle is an enzymatically operated biochemical process that requires various enzymes to work in unison. These enzymes include glutamate dehydrogenase, carbamoyl phosphate synthase I, ornithine transcarbamylase, argininosuccinate synthase, and argininosuccinate lyase.  

1. Glutamate dehydrogenase (GDH) is the catalyst for the reversible reaction of glutamate to α-ketoglutarate (α-KG), which is also known as oxidative deamination. This enzyme requires NADP as a coenzyme. 
2. Carbomoyl phosphate synthase I (CSP) catalyzes the use of two ATP molecules at a duplicated ATP-grasp fold to form carbamoyl phosphate from glutamine or ammonia and bicarbonate (HCO₃^–).
3. Ornithine transcarbamylase (OTC; EC 2.1.3.3) is present in the mitochondrial matrix and catalyzes the production of citrulline from ornithine and carbamoyl phosphate.
4. Argininosuccinate synthase (ASS; EC 6.3.4.5) catalyzes the formation of arginosuccinate from citrulline and aspartate. It is a ubiquitous enzyme in mammals and one of the rate-limiting enzymes of the ornithine cycle.   
5. Argininosuccinate lyase (ASL) is responsible for converting arginosuccinate to arginine and fumarate. Its deficiency in neonates can lead to hyperammonemia within a few days of birth, which, if left untreated, can lead to death. 
6. Arginase, a manganese metalloenzyme, catalyzes the last step of the urea cycle, where urea is formed as a byproduct and ornithine is recycled into the mitochondria for another ornithine cycle.  

Steps of Urea Cycle

The Urea Cycle is a five-step process involving amino acids and various enzymes. The catabolism of various amino acids from different cells in the body generates glutamate. The oxidative deamination of glutamate produces ammonia and alpha-ketoglutarate.

The alpha-ketoglutarate can react with other amino acids like alanine and aspartate to produce pyruvate+glutamate and oxaloacetate+glutamate, respectively. Or the alpha-ketoglutarate can enter the citric acid cycle. 

Ammonia, a highly toxic substance to the human body, especially to the brain, can lead to cerebral edema and comatose. Therefore, for its removal, our liver cells, the unsung heroes, initiate a cycle beginning from the mitochondria. 

Formation of Ammonium 

The transamination of amino acids forms glutamate. The oxidative deamination of glutamate releases ammonia (NH₃) and ɑ-ketoglutarate. The addition of hydrogen ions forms NH₄⁺ (ammonium ion). For this conversion, the required enzyme is glutamate dehydrogenase. The enzyme requires NADP (Nicotinamide adenine dinucleotide phosphate), which changes to NADPH (Nicotinamide-adenine dinucleotide phosphate). 

Conversion of Ammonium to Carbamoyl Phosphate to Enter the Urea Cycle

For the ammonium to enter the urea cycle, it undergoes a crucial conversion into carbamoyl phosphate. This conversion is catalyzed by the enzyme carbamoyl phosphate synthetase I, which plays a key role in the urea cycle. A molecule of bicarbonate (HCO₃^–) and two molecules of ATP (Adenosine Triphosphate) are also required for this conversion. The two molecules of ATP are converted into two molecules of ADP and Pi, further highlighting the energy-intensive nature of this process. Once carbamoyl phosphate forms it enters the urea cylce. 

Combination of Carbamoyl Phosphate and Ornithine

The essential amino acids ornithine and carbamoyl phosphate combine in the presence of the enzyme ornithine transcarbamoylase. Here, carbamoyl phosphate donates its carbomoyl group to ornithine to form citrulline. This reaction releases a phosphate molecule. Now, the citrulline transfers from mitochondria to the cytosol of liver cells. 

Formation of Argininosuccinate

For the formation of argininosuccinate, a crucial step in the urea cycle, a condensation reaction occurs in the cytosol of liver cells. This reaction, catalyzed by the enzyme argininosuccinate synthetase, involves the amino group of aspartate and the carbonyl group of citrulline. Notably, this reaction is energy-intensive, requiring a molecule of ATP. It also forms an intermediate production of cirtrullyl-AMP. 

Conversion to Arginine

The enzyme arginosuccinate lyase cleaves arginosuccinate, forming free arginine and a molecule of fumarate. The fumarate, an essential component, enters the mitochondria to join the pool of intermediates for the Krebs cycle. Importantly, this is the only reversible reaction in the urea cycle, highlighting the balance and regulation in this biological process.

Urea Formation

Now, the enzyme arginase cleaves arginine to yield urea and ornithine. The ornithine is transported back to mitochondria to begin another urea cycle. This reaction requires a molecule of H₂O. The urea now enters the blood and filters out as urine from the body via the kidney.  

End Products of Urea Cycle

The overall equation of the urea cycle is 

2NH₄+HCO₃+3ATP^4-+H₂O→ Urea+ 2ADP^3- +4Pi^2- + AMP^2- + 2H⁺

The urea cycle, a crucial metabolic pathway, produces urea and formate as its primary and secondary byproducts, respectively. These compounds play significant roles in various biological processes. Additionally, other byproducts of this cycle include ADP, AMP, phosphate, pyrophosphate, and water molecules. 

1. Urea: As the name suggests, the urea cycle generates urea as the main byproduct. It is released after removing ammonia from the body’s amino acids or proteins. The urea passes through the bloodstream to the kidney, where it is excreted through urine.
2. Formate: It is a secondary byproduct of the urea cycle. The fumarate is an essential intermediate of the Krebs cycle. So, it enters the mitochondrion to join the pool of intermediates. 
3. ADP, AMP, Phosphate, and Pyrophosphate: Adenosine Diphosphate (ADP), Adenosine Monophosphate (AMP), Phosphate (Pi), and pyrophosphate (PPi) are formed during the hydrolyzation of ATP (Adenosine Triphosphate). ATP is used to provide energy for the urea cycle. 
4. Water: Like ADP, AMP, Pi, and PPi water, it is also formed by the hydrolysis of ATP during the breakdown of argininosuccinate. 

Regulation of Urea Cycle

The meticulous regulation of the urea cycle is not just a biological process, but a crucial mechanism that efficiently removes excess nitrogen or ammonia. This cycle plays a pivotal role in preventing the accumulation of toxic waste (ammonia), a potential threat that can lead to ammonia toxicity, even proving lethal to the human brain. The regulation of the ornithine cycle, a key component of the urea cycle, is primarily influenced by enzyme activity or the availability of substrate. Other regulatory mechanisms, equally significant, include hormonal control. 

1. Allosteric Regulation: The ornithine cycle presents a fascinating example of allosteric regulation, a complex process that involves certain enzymes. For instance, carbamoyl protein synthase I (CPS-I), which catalyzes the first step of the cycle, is activated by N-acetyl glutamate (NAG), a positive regulator synthesized by glutamate and acetyl CoA. Similarly, the main enzyme in the cycle, ornithine transcarbamylase (OTC), is inhibited by its product, citrulline, serving as a negative feedback mechanism. This intricate interplay of enzymes and regulators is a testament to the sophistication of biological systems.
2. Hormonal Regulation: The hormones that can regulate the urea cycle include glucagon, insulin, and cortisol. Glucagon and cortisol stimulate the ornithine cycle when protein catabolism increases. Protein catabolism breaks down amino acids, producing urea as the final product. In contrast, insulin can usually inhibit the urea cycle when protein breakdown decreases. 
3. Dietary Regulation: A high protein intake provides a large number of substrates for the cycle, promoting the ornithine cycle. A lower protein intake lowers the rate of the ornithine cycle.

Functions of Urea Cycle

The urea cycle has many functions in humans, and disorders in it can be life-threatening. The functions of the ornithine cycle are as follows.

1. The ornithine cycle’s primary function is detoxifying ammonia, a highly toxic byproduct of amino acid metabolism. The potential neurological damage and other health problems caused by excess ammonia underscore the critical role of this cycle in our health. Nitrogen is an essential component, for it is a building block of protein synthesis. So, it is necessary to maintain nitrogen balance. Excess nitrogen in the body is safely removed from the body as urea. 
2. Although the ornithine cycle utilizes ATP or an energy source, it can aid in generating energy indirectly. The cycle produces fumarate, an intermediate for the citric cycle, which can help in energy production. 
3. As ammonia is slightly basic, it can contribute to the body’s acid-base balance. So, the conversion of ammonia to urea through the urea cycle helps regulate the pH of bodily fluids, ensuring they remain within a narrow, optimal range for cellular functioning.

References

1. Smith, H. Q., Li, C., Stanley, C. A., & Smith, T. J. (2019). Glutamate Dehydrogenase, a Complex Enzyme at a Crucial Metabolic Branch Point. Neurochemical research, 44(1), 117–132. https://doi.org/10.1007/s11064-017-2428-0 
2. Nelson, D., Lehninger, A., Cox, M., & Nelson, D. (2005). Lecture notebook for Lehninger principles of biochemistry, fourth edition (pp. 601-612). W.H. Freeman.