5.3: Nitrogen metabolism and the urea cycle

Amino acids play key roles as precursors to nitrogen-containing compounds (such as nucleotides and neurotransmitters), as substrates for protein synthesis, and as an oxidizable substrate for energy production (or storage). Unlike carbohydrate and lipid metabolism, we must be concerned with the fates of both the carbon- and nitrogen-containing moieties when discussing the metabolism of amino acids. In the case of amino acids, nitrogen is released as ammonia (\(\ce{NH3}\)), and at physiological pH the majority of ammonia is present as an ammonium ion (\(\ce{NH4+}\)). (It is important to note that only ammonia can cross cellular membranes.) The majority of ammonia is incorporated into urea (in the liver) and excreted by the kidney, while the remaining carbon-containing skeleton is oxidized or utilized in other anabolic pathways (i.e., gluconeogenesis).

Transport of nitrogen via amino acids

The amino acid pool is continually in flux and can be influenced by both dietary protein consumption as well as normal protein turnover within the tissues. Given that the major site of nitrogen disposal is the liver, a mechanism for transport of excess amino acid nitrogen from the peripheral tissues to the liver is in place. Both alanine and glutamine play an essential role as nontoxic carriers of ammonia from peripheral tissues to the liver (figures 5.12 and 5.13). To generate alanine and glutamine for transport, amino acids can undergo transamination reactions.

Figure 5.12: Transamination reaction.

Transamination: The movement of nitrogen

Amino transferases are a family of enzymes (which require pyridoxal phosphate; PLP) as a cofactor to help transfer nitrogen from amino acids on to keto-acid backbones. These enzymes do not free ammonia, but will transfer nitrogen from an amino group to a keto-group in an exchange or transferase reaction. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are common and clinically relevant transferases. AST will preferentially accept aspartate and transaminate it in a reaction with \(\alpha\)-ketoglutarate (the keto-acid of glutamate) to generate oxaloacetate (OAA) (the keto-acid of aspartate) and glutamate (figures 5.12 and 5.13).

Figure 5.13: Reactions catalyzed by glutamate dehydrogenase, glutaminase, and glutamine synthetase.

Glutamate dehydrogenase, glutamine synthetase, and glutaminase

In addition to transaminases, there are three other enzymes that play essential roles in nitrogen transport. Glutamate dehydrogenase (GDH) is present in most tissues and is one of the few enzymes able to fix or free ammonia. In figure 5.14, in the skeletal muscle, glutamate dehydrogenase is illustrated fixing ammonia to \(\alpha\)-ketoglutarate to generate glutamate, while in the liver it is shown freeing ammonia in the reverse reaction. The direction of the reaction will be influenced by several factors including cellular needs, the levels of NAD⁺ or NADP⁺, and levels of ammonia (figure 5.14).

Figure 5.14: Movement of ammonia from peripheral tissues to the liver.

In peripheral tissues, glutamate generated from transamination or from the GDH reaction can be used to fix an additional ammonia to generate glutamine. This reaction, catalyzed by glutamine synthetase, facilitates the synthesis and subsequent movement of excess nitrogen from peripheral tissues to the liver (figure 5.14).

In skeletal muscle, the alanine-glucose cycle is commonly used for the transport of nitrogen from the skeletal muscle to the liver. In this process, ammonia from amino acid degradation is transaminated to form glutamate. Alanine aminotransferase (AST) will transaminate glutamate with pyruvate to generate alanine (and \(\alpha\)-ketoglutarate). The alanine is released and transported to the liver where it will undergo another transamination to generate pyruvate, which is used as a substrate for glucose production (gluconeogenesis). The glucose is released from the liver and oxidized by the skeletal muscle.

The other key enzyme in nitrogen metabolism is glutaminase. Glutaminase, is active in the liver and responsible for deaminating glutamine as it is shuttled into the liver. The free ammonia can enter into the urea cycle, and the remaining glutamate can be transaminated to generate \(\alpha\)-ketoglutarate. This is in contrast to glutamine synthetase, which is primarily used by peripheral tissues as a means of generating glutamine to remove ammonia from the tissues to the liver (figure 5.14). Nitrogen metabolism, unlike glucose metabolism, is fairly consistent in the fed and fasted states. Excess dietary amino acids, which are not stored, will also require deamination, and the carbons can be stored as either glycogen or fat.

Urea cycle

Ammonia freed in the liver by glutaminase (or glutamate dehydrogenase) will readily enter the urea cycle to be incorporated into urea. A functioning urea cycle is essential for the disposal of nitrogen from catabolic processes, and if dysfunction occurs the accumulation of ammonia can be life threatening.

The urea cycle occurs in the liver and spans both the mitochondria and the cytosolic compartments. The initial free ammonia diffuses through the mitochondrial membrane and is fixed with carbon dioxide (in the form of bicarbonate) during the initial step in this process (figures 5.15 and 5.16). It is important to remember that the synthesis of urea is an anabolic process that requires ATP. Therefore deficiencies in ATP production can inhibit nitrogen disposal as well.

Figure 5.15: Overview of the urea cycle; the pathway spans both the mitochondria and cytosol.

The product of this pathway, urea, is made of two nitrogenous groups with the first coming from the free ammonia released by glutaminase. The second nitrogen is added later in the cycle by aspartate (figures 5.16 and 5.17).

Figure 5.16: Key regulatory step in the urea cycle. CPS1 is activated by N-acetylglutamate.

Figure 5.17: Entry of the second nitrogen into the urea cycle; aspartate donates the second nitrogen for the synthesis of urea.

Regulation of the urea cycle

This pathway is predominantly regulated at one key enzyme, carbamoyl phosphate synthetase 1 (figure 5.16). This enzyme requires N-acetylglutamate (NAGS) as an allosteric activator. The synthesis of NAGS is enhanced by arginine, which is an intermediate of the urea cycle. Therefore the cycle provides positive feedback on itself. As flux through the urea cycle increases, and synthesis of arginine increases, this will enhance NAGS production and increase synthesis of carbamoyl phosphate.

Unlike the other pathways discussed, the urea cycle functions independent of hormonal control as it functions to dispose of nitrogen either from excess dietary sources or from protein catabolism/turnover. In the fasted state this is especially important as the carbon skeletons produced are required as substrates for gluconeogenesis (see figure 5.3). In the fed state, amino acids can be deaminated and contribute to the carbon pool (see figures 4.12 and 4.13).

In summary, the process of nitrogen movement from the peripheral tissues to the liver is essential. It involves transamination reactions to produce alanine, and the synthesis of glutamine (by glutamine synthetase) to generate two nontoxic carriers of ammonia. Once transported to the liver, again, transamination coupled with the reactions of glutaminase and glutamate dehydrogenase will allow for ammonia to be freed and enter into the urea cycle.

Summary of pathway regulation

Metabolic pathway	Major regulatory enzyme(s)	Allosteric effectors	Hormonal effects
Urea cycle	CPS I	N-acetylglutamate (+)

Table 5.3: Summary of pathway regulation.

References and resources

Text

Ferrier, D. R., ed. Lippincott Illustrated Reviews: Biochemistry, 7th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2017, Chapter 10: Gluconeogenesis: Section II, III, IV, Chapter 11: Glycogen Metabolism: Section V, VI, Chapter 16: Fatty Acid Ketone Body and TAG Metabolism: Section III, IV, V, Chapter 19: Removal of Nitrogen from Amino Acids: Section V, VI, Chapter 23: Metabolic Effect of Insulin and Glucagon, Chapter 25: Diabetes Mellitus.

Le, T., and V. Bhushan. First Aid for the USMLE Step 1, 29th ed. New York: McGraw Hill Education, 2018, 78, 82, 86, 89–90.

Lieberman, M., and A. Peet, eds. Marks' Basic Medical Biochemistry: A Clinical Approach, 5th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2018, Chapter 3: The Fasted State, Chapter 19: Basic Concepts in Regulation, Chapter 24: Oxidative Phosphorylation and the ETC, Chapter 26: Formation of Glycogen, Chapter 28: Gluconeogenesis, Chapter 30: Oxidation of Fatty Acids, Chapter 34: Integration of Carbohydrate and Lipid Metabolism, Chapter 36: Fate of Amino Acids Nitrogen: Urea Cycle.

Figures

Grey, Kindred, Figure 5.12. Figure 5.12: Transamination reaction. 2021. CC BY 4.0. Added Liver by Liam Mitchell from the Noun Project and Muscle by Laymik from the Noun Project.

Grey, Kindred, Figure 5.13 Reactions catalyzed by glutamate dehydrogenase, glutaminase and glutamine synthetase. 2021. https://archive.org/details/5.11_20210924. CC BY 4.0.

Grey, Kindred, Figure 5.14 Movement of ammonia from peripheral tissues to the liver. 2021. https://archive.org/details/5.12_20210924. CC BY 4.0.

Grey, Kindred, Figure 5.15 Overview of the urea cycle, the pathway spans both the mitochondria and cytosol. 2021. https://archive.org/details/5.13_20210924. CC BY 4.0.

Grey, Kindred, Figure 5.16 Key regulatory step in the urea cycle. CPS1 is activated by N-acetyl glutamate. 2021. https://archive.org/details/5.14_20210924. CC BY 4.0.

Grey, Kindred, Figure 5.17 Entry of the second nitrogen into the urea cycle; aspartate donates the second nitrogen for the synthesis of urea. 2021. https://archive.org/details/5.15_20210924. CC BY 4.0.