BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E.S. Severin - 2004

CHAPTER 9. AMINO ACID METABOLISM AND FUNCTIONS

IV. Amino Acid Catabolism

Amino Acids derived from Protein Digestion and delivered to tissue Cells undergo Catabolism and anabolism, as well as specific reactions that synthesize biologically active compounds.

Catabolism of Most amino acids begins with the removal of the α-amino group. An amino acid loses its amino group through two primary types of reactions: Transamination and deamination.

A. Transamination

Transamination is The transfer of an α-amino group from an amino acid to an α-keto acid, resulting in The formation of a new keto acid and a new amino acid. The Equilibrium Constant for most of these reactions is close to unity (K_eq ~ 1.0), making the transamination process readily reversible (see Scheme A).

These reactions are catalyzed by aminotransferases (transaminases), which require Pyridoxal phosphate (PLP)—a derivative of vitamin B₆ (pyridoxine, see Chapter 3)—as a coenzyme (see Scheme B).

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Aminotransferases are located in both the Cytoplasm and Cell/35.html">Mitochondria of Eukaryotic cells. Notably, mitochondrial and cytoplasmic isoforms differ in their physicochemical properties. More than 10 different aminotransferases with distinct substrate specificities have been identified in human cells. Virtually all Amino acids can participate in transamination reactions, with the exception of Lysine, Threonine, and Proline.

1. Reaction Mechanism

Aminotransferases are classic Examples of Enzymes that catalyze reactions via a "ping-pong" mechanism (see Chapter 2). In such reactions, the first product must dissociate from the Active Site before the second substrate can bind.

The active form of aminotransferases is generated by the attachment of pyridoxal phosphate to a lysine amino group via a stable aldimine (Schiff base) linkage (Fig. 9-6). Lysine at position 258 is a component of the enzyme's active site. Additionally, ionic bonds are formed between the enzyme and pyridoxal phosphate involving charged atoms of the phosphate group and the pyridine ring nitrogen of the coenzyme.

Fig. 9-6. Binding of pyridoxal phosphate to the Active Site of an aminotransferase. The number "1" indicates the aldimine bond.

In this context, pyridoxal phosphate acts as an amino group carrier. Its aldehyde group plays the most critical role, as it can reversibly bind various amines to form Schiff bases. Transamination proceeds in two stages, during which pyridoxal phosphate undergoes reversible transformations between its free aldehyde form (PLP) and its aminated form (pyridoxamine phosphate). The reaction sequence is outlined below.

✵ In The First stage, the amino group from the first substrate (an amino acid) attaches to pyridoxal phosphate at the enzyme's active site via an aldimine bond. This yields an enzyme-pyridoxamine phosphate complex and a keto acid as the first reaction product. This process involves The intermediate formation of two Schiff bases.

✵ In the second stage, the enzyme-pyridoxamine phosphate complex binds to a keto acid (the second substrate) and, again via the intermediate formation of two Schiff bases, transfers the amino group to the keto acid. As a result, the enzyme returns to its native form, and a new amino acid is generated as the second reaction product. If the aldehyde group of pyridoxal phosphate is not occupied by a substrate amino group, it forms a Schiff base (aldimine) with the ε-amino group of a lysine side chain in the enzyme's active site (see scheme).

2. Organ-Specific Aminotransferases: ALT and AST

Transamination reactions most frequently involve amino acids whose tissue concentrations are significantly higher than those of others—namely glutamate, Alanine, aspartate, and their corresponding keto acids—α-ketoglutarate, Pyruvate, and oxaloacetate. Glutamate serves as the primary amino group donor.

Overall, these reactions can be represented by the following scheme:

The amino group acceptor for any amino acid undergoing transamination (amino acid 1) is α-ketoglutarate. Upon accepting the amino group, it is converted into glutamate, which can subsequently transfer this group to any α-keto acid to yield another amino acid (amino acid 2).

Aminotransferases exhibit substrate Specificity toward different amino acids. More than 10 distinct aminotransferases have been found in human Tissues. The most prevalent enzymes in most mammalian tissues are alanine aminotransferase (ALT), also known in the reverse reaction as glutamate-pyruvate aminotransferase (GPT), and aspartate aminotransferase (AST), also known in the reverse reaction as glutamate-oxaloacetate aminotransferase (GOT).

ALT (ALAT) catalyzes the transamination reaction between alanine and α-ketoglutarate (see Scheme A).

This enzyme is localized in the Cytosol of cells in many Organs, with the highest concentrations found in The Liver and Heart Muscle.

AST (AsAT) catalyzes the transamination reaction between aspartate and α-ketoglutarate, similarly to the previous one (see Scheme B).

Schemes A and B

This reaction yields oxaloacetate and glutamate. AST exists in both cytoplasmic and mitochondrial forms, with its highest amounts detected in the Cells of the heart muscle and liver.

Given that the highest concentrations of ALT and AST are concentrated in the liver and myocardium, while their Blood levels remain very low, these Enzymes can be considered organ-specific.

As a result of aminotransferase activity, the amino nitrogen of many amino acids is transferred to glutamate. There is good reason to believe that the accumulation of amino groups in the form of glutamic acid takes place in the cytosol. Glutamate is then transported via translocases into the mitochondria, where specific AST is active. Through the action of this enzyme, glutamate is converted back into α-ketoglutarate. The latter is utilized for the indirect Deamination of Amino acids contained within the mitochondria. This is crucial, as glutamate is the most rapidly oxidized amino acid in mammalian tissues undergoing oxidative deamination (see below).

3. Biological Significance of Transamination

Transamination reactions play a vital role in Amino acid METABOLISM. Because this process is reversible, aminotransferase enzymes function in both the catabolism and Biosynthesis OF AMINO Acids. Transamination is The final stage in the synthesis of non-Essential Amino Acids from their corresponding α-keto acids whenever the cells require them. This results in the redistribution of amino nitrogen throughout the body's tissues. Furthermore, transamination serves as the initial stage of deamination for most amino acids—that is, the starting point of their catabolism. The resulting keto acids are either oxidized in the TCA cycle or utilized for the Synthesis of glucose and Ketone Bodies. Overall, transamination does not change the total number of amino acids within The Cell.

4. Diagnostic significance of Determining Aminotransferases in Clinical Practice

In clinical practice, measuring serum AST and ALT activity is widely used to diagnose various pathologies.

Under normal conditions, The activity of these enzymes in the blood is very low, ranging from 5 to 40 U/L. When cells of the respective organ are damaged, the enzymes leak into the bloodstream, where their activity increases sharply. Since AST and ALT are most active in the cells of the liver, heart, and, to a lesser extent, skeletal Muscles, they are used to diagnose diseases affecting these organs (see Section 2). In heart muscle cells, The amount of AST significantly exceeds that of ALT, whereas the reverse is true in the liver. Therefore, simultaneously measuring the activity of both enzymes in blood serum is particularly informative. The ratio of AST to ALT activities is referred to as the De Ritis ratio. Normally, this ratio is 1.33 ± 0.42. In myocardial infarction, blood AST activity increases 8- to 10-fold, while ALT activity rises 1.5- to 2.0-fold. AST activity increases most dramatically during tissue necrosis, as both the cytoplasmic and mitochondrial forms of the enzyme are released into the blood. Consequently, the De Ritis ratio rises sharply during a myocardial infarction.

In hepatitis, serum ALT activity increases approximately 8- to 10-fold compared to the normal range, while AST increases 2- to 4-fold, causing the De Ritis ratio to drop to 0.6. However, in liver cirrhosis, this ratio increases, indicating cellular necrosis where both forms of AST are released into the bloodstream.

B. Deamination of Amino Acids

AMINO ACID DEAMINATION is the process by which the α-amino group is cleaved from an amino acid, yielding the corresponding α-keto acid (a nitrogen-free residue) and releasing an ammonia molecule. Subsequent Metabolic pathways of these deamination products are illustrated in Fig. 9-7.

Fig. 9-7. Metabolic Fate of amino acid deamination products.

Ammonia is toxic to the Central Nervous system (CNS); therefore, in humans and mammals, it is converted into urea, a non-toxic and highly soluble compound. Ammonia is eliminated from the body in the form of urea and ammonium salts. The nitrogen-free carbon Skeleton is utilized for Amino acid synthesis via transamination, in Gluconeogenesis and ketogenesis, in anaplerotic reactions to replenish depleted TCA cycle intermediates, and in oxidation reactions yielding CO₂ and H₂O.

Several PATHWAYS OF AMINO acid deamination exist:

✵ oxidative;

✵ indirect (transdeamination);

✵ non-oxidative;

✵ intramolecular.

1. Oxidative Deamination

The deamination of glutamic acid occurs most actively in tissues. The reaction is catalyzed by the enzyme Glutamate dehydrogenase, and NAD⁺ serves as its coenzyme. The reaction proceeds in two stages. First, enzymatic dehydrogenation of glutamate takes place with the formation of α-iminoglutarate, followed by non-enzymatic hydrolytic Cleavage of the imino group as ammonia, yielding α-ketoglutarate (see the scheme below).

Oxidative deamination of glutamate is a reversible reaction and, upon an increase in intracellular ammonia concentration, can proceed in the reverse direction as reductive amination of α-ketoglutarate.

Glutamate dehydrogenase is highly active in the mitochondria of cells across virtually all organs, except muscles. This enzyme is an oligomer consisting of 6 subunits (molecular weight 312 kDa). Glutamate dehydrogenase plays a crucial role as a regulatory enzyme in amino acid metabolism. Allosteric inhibitors of glutamate dehydrogenase (ATP, GTP, NADH) induce enzyme dissociation and loss of glutamate dehydrogenase activity. High concentrations of ADP activate the enzyme. Thus, a low cellular energy status stimulates The breakdown of Amino Acids and the formation of α-ketoglutarate, which enters the TCA cycle as an energy substrate. Glutamate dehydrogenase can be induced by Steroid Hormones (cortisol).

L-Amino Acid Oxidase

The enzyme L-amino acid oxidase, capable of deaminating certain L-amino acids, has been found in the liver and Kidneys (see the scheme at the end of the page).

FMN acts as the coenzyme in this reaction. However, THE CONTRIBUTION OF L-amino acid oxidase to deamination is apparently negligible, as its pH optimum lies in the alkaline range (pH 10.0). In cells where the physiological pH is close to neutral, the enzyme's activity is very low.

D-Amino Acid Oxidase is also found in the kidneys and liver. It is an FAD-dependent enzyme. The pH optimum for this oxidase lies in the neutral range, making the enzyme more active than L-amino acid oxidase. The Physiological Role of D-amino acid oxidase is minor, since the quantity of D-isomers in the body is extremely small—given that dietary and tissue Proteins in humans and animals contain exclusively natural L-amino acids. Presumably, D-amino acid oxidase facilitates their conversion into the corresponding L-isomers (Fig. 9-8).

Fig. 9-8. Biological Role of D-amino acid oxidase.

2. Indirect Deamination (Transdeamination)

Most amino acids cannot be deaminated in a single step unlike Glu. The amino groups of such Amino acids are transferred via transamination to α-ketoglutarate to form glutamic acid, which subsequently undergoes direct oxidative deamination. This two-stage mechanism of amino acid deamination is termed transdeamination, or indirect deamination:

Indirect deamination of amino acids proceeds with the participation of two enzymes: aminotransferase (coenzyme PLP) and glutamate dehydrogenase (coenzyme NAD⁺).

The Significance of these reactions in amino acid metabolism is immense, as indirect deamination is the primary pathway for the deamination of most amino acids. Both stages of indirect deamination are reversible (Fig. 9-9), ensuring both Amino Acid Catabolism (Fig. 9-9, A) and the potential synthesis of virtually any amino acid from its corresponding α-keto acid (Fig. 9-9, B).

Fig. 9-9. Biological role of indirect deamination. A — during catabolism, nearly all Natural Amino Acids first transfer their amino group to α-ketoglutarate via transamination, yielding glutamate and the corresponding keto acid. Glutamate is then subjected to direct oxidative deamination catalyzed by glutamate dehydrogenase, producing α-ketoglutarate and ammonia; B — when amino acid synthesis is required and appropriate α-keto acids are available, both stages of indirect deamination proceed in reverse. Reductive amination of α-ketoglutarate yields glutamate, which enters into transamination with the corresponding α-keto acid, resulting in the synthesis of a new amino acid.

In Muscle tissue, the activity of glutamate dehydrogenase is low; therefore, during intensive physical exertion, these cells utilize an alternative pathway of indirect deamination involving the IMP-AMP cycle. Initially, the amino group is transferred from amino acids to aspartate, then to inosinic acid (IMP), and finally, AMP is deaminated. The presented scheme illustrates The sequence of reactions in indirect Non-Oxidative Deamination:

Four Stages of the process can be distinguished:

✵ transamination with α-ketoglutarate, forming glutamate;

✵ transamination of glutamate with oxaloacetate (enzyme AST), forming aspartate;

✵ transfer of the amino group from aspartate to IMP (inosine monophosphate), forming AMP and fumarate;

✵ hydrolytic deamination of AMP.

The transfer of the amino group from aspartate and the synthesis of AMP proceed as follows (see scheme A).

The deamination of adenylic acid is catalyzed by the enzyme AMP deaminase (see Scheme B).

Scheme A

Scheme B

This pathway of deamination predominates in muscles during intense exertion, which leads to the accumulation of lactic acid. The released ammonia prevents intracellular acidification caused by lactate production.

3. Non-oxidative deamination

Human liver contains specific enzymes that catalyze the non-oxidative deamination of the amino acids Serine, threonine, and Histidine.

Non-oxidative deamination of serine is catalyzed by serine dehydratase (see Scheme A).

The reaction begins with the elimination of a Water molecule and the formation of a methylene group, followed by a non-enzymatic molecular rearrangement that yields an imino group weakly bound to the α-carbon atom. Subsequent non-Enzymatic Hydrolysis releases an ammonia molecule to form pyruvate.

Non-oxidative deamination of threonine is catalyzed by the enzyme Threonine dehydratase. The reaction mechanism is analogous to the deamination of serine (see Scheme B on p. 477).

These enzymes are pyridoxal phosphate-dependent.

Non-oxidative deamination of histidine, mediated by histidase (histidine ammonia-lyase), is an intramolecular process, as the ammonia molecule is formed from The amino acid's own atoms without the participation of a water molecule. This reaction takes place exclusively in the liver and Skin (see Scheme C).

Conditions such as cystinuria, Hartnup disease, and several others arise from defects in neutral amino acid transporters in the intestine and kidneys. A congenital disorder associated with a defect in the enzyme 5-oxoprolinase has also been described (Fig. 9-5, reaction 4), resulting in the urinary excretion of oxoproline. Affected individuals exhibit impaired Amino Acid Transport into tissues and disrupted cellular metabolism.