BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 6. AMINO ACID METABOLISM AND FUNCTIONS. PROTEIN BIOSYNTHESIS

6.4. Amino Acid Catabolism

6.4.1. 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 Amino Acid and a new keto acid. The Equilibrium Constant for most of these reactions is close to one (K_eq~1.0), making the amination process reversible:

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The reaction is catalyzed by aminotransferases (transaminases), Enzymes that utilize Pyridoxal phosphate (PLP), a derivative of vitamin B₆, as a coenzyme:

Reaction Mechanism. Aminotransferases are found in both the Cytoplasm and Cell/35.html">Mitochondria of Eukaryotic Cells. Furthermore, mitochondrial and cytoplasmic forms of these enzymes differ in their physicochemical properties. Human cells contain approximately 10 different aminotransferases that vary in substrate Specificity. Virtually all Amino Acids can participate in transamination reactions, with the exceptions of Lysine, Threonine, and Proline.

Aminotransferases are a classic example of enzymes that catalyze reactions via a "ping-pong" mechanism. In such reactions, the first product must leave the Active Site before the second substrate can bind to it.

The active form of aminotransferases is generated through the attachment of pyridoxal phosphate to the amino group of a lysine residue via a stable aldimine (Schiff base) linkage (Fig. 6.6). 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 the charged atoms of the phosphate group and the nitrogen atom in the pyridine ring of the coenzyme.

Fig. 6.6. Attachment of pyridoxal phosphate to the Active Site of an aminotransferase: "1" indicates the aldimine linkage

In this context, pyridoxal phosphate acts as an amino group carrier. Its aldehyde group plays a crucial role by reversibly binding various amines to form Schiff bases. Transamination reactions occur in two stages, during which pyridoxal phosphate undergoes reversible transformations between its free aldehyde form (pyridoxal phosphate) and its aminated form (pyridoxamine phosphate).

The sequence of transamination reactions consists of two stages:

✵ In The First stage, the amino group from the first substrate (an amino acid) is attached to pyridoxal phosphate at the enzyme's active site via an aldimine linkage. This yields an enzyme-pyridoxamine phosphate (PMP) 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 combines with a keto acid (the second substrate) and, again through 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:

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

The overall reaction can be represented by the following scheme:

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

Aminotransferases exhibit substrate specificity toward various amino acids. Human Tissues contain over 10 different aminotransferases. The most prevalent enzymes in most mammalian tissues are alanine aminotransferase (ALT), also referred to in the reverse reaction as glutamate-pyruvate aminotransferase (GPT), and aspartate aminotransferase (AST), referred to in the reverse reaction as glutamate-oxaloacetate aminotransferase (GOT).

ALT (ALAT) catalyzes the transamination reaction between alanine and α-ketoglutarate:

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

AST (SGOT) catalyzes the transamination reaction between aspartate and α-ketoglutarate, similarly to the previous one:

This reaction yields oxaloacetate and glutamate. AST exists in both cytoplasmic and mitochondrial forms, with the highest levels found in heart muscle and liver cells.

Thus, the greatest amounts of ALT and AST are concentrated in The Liver and myocardium, while their Blood levels remain very low, indicating a high degree of organ specificity.

Through the action of aminotransferases, the amino nitrogen of many amino acids is transferred to glutamate. There is strong evidence that amino groups accumulate in the form of glutamic acid within the cytosol. Glutamate is then transported via translocases into the mitochondria, where active, specific AST is present. As a result of this enzyme's activity, glutamate is converted back into α-ketoglutarate, which is subsequently utilized for the indirect Deamination of Amino acids located within the mitochondria. This is crucial because glutamate is the only amino acid in mammalian tissues capable of undergoing rapid Oxidative Deamination.

Biological Significance of transamination. Transamination reactions play a vital role in Amino acid METABOLISM. Since the process is reversible, aminotransferase enzymes function in both the Catabolism and Biosynthesis OF AMINO Acids. Transamination serves as the final step 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 body tissues. Furthermore, transamination constitutes the initial stage of deamination for Most amino acids, marking the starting point of their catabolism. The resulting keto acids are either oxidized in the TCA cycle or used for the Synthesis of glucose and Ketone Bodies. Notably, transamination does not alter the total pool of amino acids within The Cell.

In clinical practice, determining serum AST and ALT activity is widely used for diagnosing various diseases. Under normal conditions, The activity of these enzymes in the blood is very low (5-40 IU/L). When cells of a target organ are damaged, these enzymes leak into the bloodstream, causing their activity to rise sharply. Because AST and ALT are most active in the Cells of the liver, heart, and to a lesser extent, skeletal Muscles, they serve as key diagnostic markers for pathologies in these organs. In heart muscle cells, The amount of AST significantly exceeds that of ALT, whereas the reverse is true in the liver. Consequently, simultaneously measuring the activity of both enzymes in blood serum holds significant diagnostic value. The ratio of AST to ALT activity is known as the de Ritis ratio. Normally, this ratio is 1.33 ± 0.42. In myocardial infarction, blood AST activity increases 8-10 fold, while ALT activity rises 1.5-2.0 fold. AST activity surges most dramatically during tissue necrosis because both the cytoplasmic and mitochondrial forms of the enzyme are released into the blood. In cases of myocardial infarction, the de Ritis ratio increases sharply.

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