18.2: Metabolic Fates of Amino Groups

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Learning Goals (ChatGPT o3-mini)

1. Protein Digestion and Amino Acid Liberation:

   • Describe the stepwise breakdown of dietary proteins beginning in the stomach (via pepsin) and continuing in the small intestine (via trypsin, chymotrypsin, carboxypeptidases, and aminopeptidases), leading to the formation of free amino acids and small peptides.
   • Explain how the acidic environment of the stomach aids in protein unfolding and facilitates enzymatic cleavage.

2. Proteasomal Degradation and Amino Acid Recycling:

   • Explain the role of the proteasome and ubiquitin tagging in degrading intracellular proteins, and how this process contributes to the amino acid pool available for metabolism.

3. Integration of Amino Acid Catabolism into Central Metabolism:

   • Summarize how different amino acids are catabolized to yield key intermediates (e.g., pyruvate, acetyl-CoA, oxaloacetate, and α‑ketoglutarate) that feed into glycolysis and the citric acid cycle.
   • Differentiate between glucogenic and ketogenic amino acids and discuss their respective roles in energy production and biosynthesis.

4. Transamination and PLP-Dependent Reactions:

   • Describe the mechanism of transamination reactions, including the role of pyridoxal phosphate (PLP) as a cofactor in facilitating the reversible transfer of amino groups.
   • Compare the functions of aspartate transaminase (AST/GOT) and alanine transaminase (ALT/GPT) in linking amino acid metabolism with carbohydrate metabolism.

5. Glutamate and Glutamine in Nitrogen Metabolism:

   • Explain how glutamine and glutamate serve as key nitrogen carriers in the body, facilitating the safe transport of ammonia (NH₃/NH₄⁺) and preventing its toxic accumulation.
   • Describe the role of glutaminase in converting glutamine to glutamate and the function of glutamate dehydrogenase in oxidative deamination to form α‑ketoglutarate and release ammonium.

6. The Glucose-Alanine Cycle:

   • Illustrate the glucose-alanine cycle, detailing how alanine is formed in muscle via transamination, transported to the liver, and then converted back to pyruvate for gluconeogenesis.
   • Contrast the glucose-alanine cycle with the Cori cycle, noting the different substrates and metabolic fates involved in each.

7. Regulation and Toxicity of Ammonia:

   • Discuss why high concentrations of ammonia are toxic to cells and the biochemical strategies used to mitigate this toxicity, such as incorporation into glutamine.
   • Explain how the liver processes ammonia, either by converting it to urea, incorporating it into amino acids, or recycling it for biosynthesis.

8. Integration of Nitrogen Metabolism with Energy Homeostasis:

   • Analyze how amino acid catabolism interconnects with central energy metabolism, emphasizing how the breakdown of amino acids supplies intermediates for ATP production and biosynthetic pathways.
   • Evaluate the physiological relevance of amino acid catabolism in different nutritional states (fed, fasting, or diabetic conditions).

By achieving these learning goals, students will gain a comprehensive understanding of how amino acids are derived, metabolized, and integrated into central metabolic pathways, as well as the regulatory mechanisms that ensure efficient nitrogen handling and energy homeostasis in living organisms.

From the previous section on the nitrogen cycle, it should be clear that NH₃/NH₄⁺ has a central role in metabolism. Nature's plant fertilizer is ammonium derived from bacterial nitrogenase and the human-derived Born-Haber process. Animals get their ammonia mostly from ingested plants (primary producers) and animal protein. In vertebrates, proteins get digested into small proteins and peptides, starting in the stomach where pepsin cleaves proteins after aromatic and Leu side chains into smaller fragments. The low pH in the stomach (1-2) facilitates protein unfolding, which allows great access to buried side chains for pepsin cleavage. As the ingested materials move into the small intestine, the protein fragments are further cleaved into small peptides by trypsin and chymotrypsin and then into individual amino acids by carboxypeptidases (which cleave C-terminal amino acids) and aminopeptidases (which cleave at the N-terminal end). The individual amino acids are then adsorbed (along with di- and tripeptides) into the epithelial cells lining the small intestine. Peptidases cleave short peptides, and amino acids are moved into the blood through cell membrane transporters, which are coupled to sodium ion intake. Amino acids are taken up by tissues for use in protein synthesis and by the liver of vertebrates for metabolic processing. The gut proteolytic enzymes are secreted into the gut lumen as precursors (proenzymes), where they are activated either autocatalytically or by other proteases into their mature form.

Amino acids are also derived from the degradation of cellular proteins by a supramolecular assembly called the proteasome. Proteins designated for cleavage are covalently modified by ubiquitin, a short ubiquitous protein, which targets them to the proteasome.

Degradation of amino acids to produce ammonium

Here is some good advice on giving a seminar: tell what you will tell them, tell them, and tell them what you told them. These words imply that repetition is one of the keys to learning. So look at Figure \(\PageIndex{1}\), which shows an overall view of the glycolytic and TCA pathways, and see how amino acid catabolism fits into what you already know!

Figure \(\PageIndex{1}\): Amino acids and their involvement with the glycolytic and TCA pathways

A summarizing principle is that amino acids degrade to form intermediates that fit into these two common pathways and can be used for ATP production and biosynthesis. Those that form pyruvate are glucogenic amino acids, while those that form acetyl-CoA are ketogenic (form ketones and could form ketone bodies). Many form direct intermediates in the TCA cycle.

This chapter will focus on glutamine/glutamic acid, alanine (which forms pyruvate), and aspartic acid. In this section, we will focus on the role of amino acid catabolism in producing ammonium. When new students confront the structures, properties, and reactions of amino acids, they are undoubtedly daunted. Even biochemists would be if amino acid metabolism is outside their active research area. Perhaps some interesting biological properties of free amino acids might pique your interest. For example, did you know that ...

• glutamate and glutamine are the most abundant amino acids in red blood cells;
• alanine and glutamine are the most abundant amino acids in the body;
• glutamic acid, glutamine, and alanine are the most abundant amino acids in the cerebrospinal fluid CSF (50-55% of total amino acids);
• glutamine is the most abundant amino acid in blood serum;
• In contrast, leucine is the most abundant amino acid in proteins, and free leucine is a prime regulator of protein synthesis through its interaction with the mTOR kinase complex. The other top three amino acids in proteins are serine, lysine, and glutamic acid.

From the above list, Glu and Gln appear to play special roles in metabolism and signaling. They also play major roles in ammonium metabolism as the pair is a major source of NH₄⁺ production in cells. Gln has two Ns (an amine and an amide), so it's not an unexpected source of nitrogen:

• Glutamine can give up its amide nitrogen to form NH₄⁺ on conversion to glutamic acid, a reaction catalyzed by glutaminase
• Glutamic acid can undergo oxidative deamination to form α-ketoglutarate (a TCA intermediate) and free NH₄⁺, a reaction catalyzed by glutamate dehydrogenase
• Glutamic acid can also give up its ammonia nitrogen to a ketoacid like pyruvate to form α-ketoglutarate (a TCA intermediate) and another amino acid (alanine if pyruvate was used as the keto acid), a reaction catalyzed by a class of enzymes called transaminases (makes sense again), which are also called aminotransferases. In this case, free NH₄⁺ is not formed but is passed to a keto acid to form another amino acid.

In vertebrates, free amino acids are metabolized in the liver. Amino acids that enter the liver transfer their ammonia group to α-ketoglutarate (aka 2-oxoglutarate) to form glutamic acid, which enters the mitochondria and can be cleaved by glutamate dehydrogenase to form α-ketoglutarate and free ammonium. The NH₄⁺ produced is either recycled or excreted as NH₄⁺ in fish, urea, H₂N(C=O)NH₂ in vertebrates, and uric acid in birds and reptiles. Excess amino acids (which again can't be stored for energy) in other organs pass their ammonia group to glutamic acid to form glutamine, which then heads to the liver for processing. Glutamine becomes a safe way to transfer 1-2 ammonium equivalents through the blood.

Alanine and aspartic acid also play secondary roles. Ala is one transamination step away, through the removal of its amine group, from pyruvate. Pyruvate is a crucial α-ketoacid end-product of glycolysis and entry reactant for the TCA cycle (after pyruvate dehydrogenase). In muscle, excess amino acids pass their ammonia groups to pyruvate to form alanine, another "safe" carrier of ammonium, which heads to the liver for processing. Likewise, aspartic acid is one transamination step away, through removing its amino group, to form oxaloacetate, another TCA α-ketoacid.

Figure \(\PageIndex{2}\) shows the key α-keto acids, which are also intermediates of the TCA cycle or feed into it (pyruvate) and their respective transamination amino acid products. Their structures immediately show a link between amino acid and carbohydrate metabolism.

 

Figure \(\PageIndex{2}\): α-ketoacids and their transamination amino acid products

We will focus most of our attention on glutamine and glutamic acid. Figure \(\PageIndex{3}\) summarizes the important enzymatic steps in the conversion of Gln ↔ Glu ↔ α-KG

Figure \(\PageIndex{3}\): Important enzymatic steps in the conversion of Glutamine ↔ Glutamic acid ↔ α-ketoglutarate

Pyridoxal phosphate and transamination reactions

Most free amino acids start their metabolic degradation in the liver with transamination reactions, using PLP as a cofactor. PLP enables other biochemical reactions of amino acids, including racemizations, decarboxylation, and dehydration (of serine). PLP is covalently attached to a Lys through a Schiff base linkage in the enzyme during the reaction cycle, so it is not considered a substrate but rather a cofactor that returns to its original state after the reaction.

Figure \(\PageIndex{4}\) shows the structure of PLP and the imine formed on reaction with an amino acid. The reaction is readily reversible through hydrolysis, which would presumably not be present in the active site of PLP-dependent enzymes. A lysine amine group in the enzyme forms a covalent adduct with PLP to form an imine. The imine in the figure below is called an internal aldimine. Internal implies that the source of the N in the imine link is a Lys internal to the protein. If you replace the N in the imine with an O, the functional group with the C=O would be an aldehyde. Hence, the imine shown is an aldimine.

Figure \(\PageIndex{4}\): PLP and its imine-protein conjugate

William Jencks, in his classic text, Catalysis in Chemistry, describes the mechanistic beauty of PLP-dependent enzymes:

"It has been said that God created an organism especially adapted to help the biologist find an answer to every question about the physiology of living systems; if this is so, it must be concluded that pyridoxal phosphate was created to provide satisfaction and enlightenment to those enzymologists and chemists who enjoy pushing electrons, for no other coenzyme is involved in such a wide variety of reactions, in both enzyme and model systems, which can be reasonably interpreted in terms of the chemical properties of the coenzyme. Most of these reactions are made possible by a common structural feature. That is, electron withdrawal toward the cationic nitrogen atom of the imine and into the electron sink of the pyridoxal ring from the alpha carbon atom of the attached amino acid activates all three of the substituents of this carbon for reactions which require electron withdrawal from this atom."

When PLP-dependent enzymes react with an external amino acid acting as a substrate, the amine of the incoming amino acid replaces the amine from the enzyme's active Lys to produce an external imine. The figure below shows an external amino acid in imine linkage to PLP, and in the form shown is an aldimine.

Each PLP-dependent reaction involves a protonated and positively charged pyridinium N as an electron sink, facilitating cleavage of each bond around the C_α of the covalently attached amino acid. Bond cleavage would leave a lone pair and negative charge on the α-carbon and no place to stabilize it by resonance. On forming a Schiff base with a pKa of around 7.0, the imine nitrogen, especially in its protonated form, is an excellent stabilizer of the negative charge. Continued delocalization of the lone pair and negative charge to the positive pyridinium nitrogen further facilitates the stabilization and, hence, the reaction. Figure \(\PageIndex{5}\) shows the cleavage of bonds around the C_α of an amino acid-PLP Schiff base.

Figure \(\PageIndex{5}\): Cleavage of bonds around the C_α of an amino acid-PLP Schiff base

aldimines and ketimines

We will encounter two similar types of structures, aldimines and ketimines, when we discuss the chemistry of PLP.  They look very similar but are named differently, so it's easy to confuse them.  Here they are:

Aldehyde/aldimine and ketone/ketimine structures

It's easy to differentiate them by looking at the C atom connected to a =O or =NR.  If the C atom has an R and H group attached, it's an aldehyde or aldimine. It is a ketone or a ketimine if it has 2 R groups attached.

Figure \(\PageIndex{6}\) shows the mechanism for a transamination reaction using PLP as a cofactor. This is, of course, the reaction most relevant to this chapter section. The amino acid substrate is first shown in an aldimine linkage to PLP. Also, note the conversion of an aldimine to a ketimine in the next step.

Figure \(\PageIndex{6}\): Mechanism for a transamination reaction using PLP as a cofactor

The model below shows the active site of aspartate transaminase from E. Coli (pdb code 1aam) with PLP in Schiff base linkage with lysine 258. Three amino acids critical for enzyme function (Trp 142, Asp 223, and Lys 258) and all side chains within 5 angstroms from the active site defined by those amino acids are shown. Lys 258 forms the Schiff base with PLP cofactor and acts as a general acid/base on interconversion between the aldimine and ketamine forms (see figure above). The ring of Trp 142 forms pi-stacking interactions with the aromatic ring of PLP and helps position it. Asp 223 acts as a general acid/base in the catalytic cycle, facilitating the deprotonation of the amine group of the substrate Asp, making it a potent nucleophile in forming the external aldimine.

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the active site of aspartate transaminase from E. Coli with PLP (1aam)

Figure \(\PageIndex{7}\): Active site of aspartate transaminase from E. Coli with PLP (1aam). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...YeQ6kah4EuERT9

The various names and abbreviations for transaminases/aminotransferases (which are reversible) can be confusing. Two key ones whose levels are used to assess liver toxicity in clinical tests are AST (GOT) and ALT (GPT).

• ASpartate Amino Transferase (AST) is the same enzyme as Glutamate Oxalacetate Transaminase (GOT), named for the reverse reaction:

aspartate + α−ketoglutarate ↔ oxalacetate + glutamate

• ALanine Amino Transferase (ALT) is the same enzyme as Glutamate Pyruvate Transaminase (GPT), named for the reverse reaction:

alanine +α−ketoglutarate ↔ pyruvate + glutamate

We will consider other reactions of PLP-dependent enzymes when needed.

To summarize, different amino acids in the liver can donate their amine groups to α-ketoglutarate to form glutamic acid in a reaction catalyzed by a transaminase/aminotransferase.

Production of NH₃ - Glutamate Dehydrogenase

Now that we have made glutamic acid from various amino acids, we can break it down to make α-ketoglutarate and ammonium again. The overall reversible reaction catalyzed by the enzyme glutamate dehydrogenase in mitochondria is shown in Figure \(\PageIndex{8}\). Mammalian livers can also use NADP⁺ as an oxidizing agent.

Figure \(\PageIndex{8}\): Overall reversible reaction catalyzed by the enzyme glutamate dehydrogenase in mitochondria

Humans have two isozymes. GluDH1 is expressed at high levels in the liver, brain, pancreas, and kidney, while GluDH2 is found in the retina, testes, and brain. GluDH1 can use both NAD⁺ and NADP⁺ in both anabolic and catabolic reactions.

The reaction/product pairs again show this reaction as a link between protein and carbohydrate metabolism. Figure \(\PageIndex{9}\) shows just the first step in the glutamate dehydrogenase reaction.

Figure \(\PageIndex{9}\): First step in the reaction of glutamate dehydrogenase

The C-N in Glu becomes C=N in the imine intermediate. You should recognize this as an oxidation step even if the obvious oxidizing agent, NAD(P)⁺, were not shown. That should also be evident since a hydride (H^-) with a lone pair is removed from glutamic acid, not an H⁺ as in an acid/base reaction. Subsequent steps include nucleophilic attack by water (enhanced by a proximal Lys acting as a general base) on the imine C, followed by ammonia release on alpha-ketoglutarate formation. The overall reaction is an oxidative deamination.

NH₃/NH₄⁺ are toxic in high concentrations. One possible reason is that the NH₄⁺ cation might compete with the transport of other ions across neural membranes, altering transmembrane potentials and, hence, neural function. Given this toxicity, you would expect that glutamate dehydrogenase is highly regulated and is in mammals. The mammalian enzyme is a hexamer of identical subunits, suggesting allosteric regulation of activity (much like tetrameric hemoglobin). ADP and leucine are allosteric activators, while GTP, palmitoyl CoA, and ATP act as inhibitors. The GTP/ADP regulation is consistent with the notion that the enzyme is regulated by the need for cellular energy (remember that GTP is formed in the TCA cycle).

Although this reaction is written above in the direction of NH₃ formation, the reaction is reversible, even though the ΔG⁰=-6.2 kcal/mol (-26 kJ/mol) and though the human enzymes have a high Km for ammonia (12-62 mM, Brenda Database). As the pH decreases from 8 to 7, the Km for ammonia increases from around 12 to 60 mM. This implies that on glutamate uptake and in acidotic conditions, the reaction runs exclusively in the direction of NH₃ release - as an oxidative deamination reaction.

Transporting ammonium equivalents in the blood - Glutamine and Alanine

NH₃/NH₄⁺ are toxic in high concentrations, so mechanisms must be used to transport it in the blood. Ideally, it would be transported in a nontoxic form. Glutamic acid is a key metabolic molecule, so glutamine is the preferred molecule for safe "NH₃/NH₄^+" transport from tissue to the liver, while alanine is the choice for muscle tissue. This less-toxic mode of transport is similar in principle to using soluble ketone bodies to transport the energy equivalent of the less soluble fatty acids in the blood.

A key enzyme for this process is glutamine synthetase, which catalyzes this reaction:

NH₄⁺ + ATP + Glu → Gln + ADP + HPO₄^2- + H⁺

This reaction is important as most ingested glutamine is converted to glutamate on absorption.

NH₃/NH₄⁺ made in the intestine and kidneys enters the circulation, where it heads to the liver to eventually produce urea.

Figure \(\PageIndex{10}\) shows the Glucose-Alanine Cycle compared to the Cori cycle we discussed in a previous chapter.

Figure \(\PageIndex{10}\): Glucose-Alanine Cycle in comparison to the Cori cycle

Production of NH₃ - Glutaminase

Most amino acids arriving in the liver for degradation go through two enzymes, transaminases, to form glutamic acid, followed by glutamate dehydrogenase to form a TCA intermediate (alpha-ketoglutarate) and ammonia. Glutamine carries excess ammonia in the bloodstream (see below). When it arrives in the liver, it can lose its amide NH₂ as ammonium as it is converted to glutamic acid by the enzyme glutaminase. This nonoxidative enzyme is expressed in the liver, brain, and kidney. Some glutamates can lose ammonia through the enzyme glutamate dehydrogenase, but most are reserved for protein synthesis or creating anabolic precursors.

As mentioned above, glutamine is the most abundant free amino acid in the body and cells. It can donate its amide N through reactions catalyzed by amidotransferase (not to be confused with aminotransferase), incorporating nitrogen into many biomolecules.

Metabolic Summary

• Glutamine is both a carrier of ammonia and a carbon backbone used in metabolism when converted to α-ketoglutarate. It can be metabolized for energy and used in biosynthesis for nucleotides and neurotransmitters. Its levels are controlled by glutamine synthase and glutaminase. When we get to synthetic pathways, you will find that glutamine is a NH₃/NH₄⁺ donor for synthesizing other amino acids and nucleotides, amino sugars, and NAD⁺.
• Glutamine absorbed in the intestines is mostly converted to glutamic acid in the intestinal epithelial cells and transported by the blood. The rest heads to the liver for processing. Hence, most glutamine must be synthesized by glutamine synthase, a cytosolic enzyme found in most mammalian cells. However, it is most abundant in muscle, liver, and adipose cells, which express little glutaminase and from which it can be exported. Glutaminase in the liver and kidney leads to ammonium production.
• Glutamine is a source of energy and carbon for biosynthesis by tumor cells, which need energy and intermediates for synthesis by rapidly proliferating cells. Aerobic glycolysis ("Warburg effect") allows that to occur.

Summary

This chapter explores the metabolic fate of proteins and amino acids, emphasizing how nitrogen is managed and integrated into central metabolism. It begins by detailing the process of protein digestion, which starts in the acidic environment of the stomach and continues in the small intestine with enzymes such as trypsin, chymotrypsin, carboxypeptidases, and aminopeptidases. These enzymes break proteins into peptides and amino acids, which are then absorbed through the intestinal epithelium.

In addition to dietary protein breakdown, the chapter describes the intracellular degradation of proteins by the proteasome. Proteins destined for degradation are tagged with ubiquitin, directing them to the proteasome where they are systematically cleaved into amino acids.

The catabolism of these amino acids is integrated into central metabolic pathways. Transamination reactions—catalyzed by PLP-dependent enzymes such as aspartate transaminase (AST/GOT) and alanine transaminase (ALT/GPT)—transfer amino groups to ketoacids, forming key intermediates like oxaloacetate and pyruvate. These reactions not only facilitate the use of amino acids for energy production via the TCA cycle but also play a critical role in biosynthesis.

Glutamate and glutamine are highlighted as central players in nitrogen metabolism. Glutaminase converts glutamine to glutamate, and glutamate dehydrogenase then catalyzes the oxidative deamination of glutamate to produce α‑ketoglutarate and ammonium. Because free ammonia (NH₃/NH₄⁺) is toxic, the body employs safe transport mechanisms—using glutamine and alanine as non-toxic carriers—to shuttle nitrogen from peripheral tissues to the liver, where ammonia is ultimately converted to urea for excretion.

By linking protein digestion, intracellular proteolysis, transamination, and deamination processes, this chapter illustrates how amino acid catabolism is seamlessly integrated with carbohydrate metabolism to support both energy production and biosynthetic demands. Overall, the chapter underscores the delicate balance of nitrogen metabolism and its vital role in maintaining cellular and systemic homeostasis.