General Reactions of Amino Acids | Biochemistry

In this article we will discuss about the two main types of general reactions in amino acids, i.e, enzymatic reactions using pyridoxal phosphate as the co-enzyme and deamination.

1. Enzymatic Reactions using Pyridoxal Phosphate as the Co-Enzyme:

A number of reactions involving amino acids, like transamination, decar­boxylation, elimination of the side chain with maintenance of the α-amino-car- boxylic group (e.g., transformation glycine ←→ serine), elimination of H₂O or H₂S (for example, under the influence of serine-dehydratase or cysteine desulphydrase), racemization (e.g., L-alanine ←→ D-alanine) require the same coenzyme: pyridoxal phosphate.

The fact that the reactions catalyzed are very different, while the coenzyme is identical, shows the decisive influence of the apoenzyme (the protein part) on the evolution of the reaction. However, as will be seen for the first three reactions mentioned above, the same inter­mediate product is formed (a Schiff base), then the reactions proceed in different directions (see fig. 7-1).

One must indeed imagine pyridoxal phos­phate in space as a plane molecule (aromatic cycle). During the formation of the intermediate Schiff base, the a-carbon and nitrogen of the amino acid are placed in the plane of the coenzyme.

Then the enzyme will force one of the three other bonds of the a-carbon of the amino acid to take a position perpen­dicular to this plane and will use the electron attractive power of the protonated coenzyme (>N⁺H) to attract on the α-carbon the electronic doublet of this bond. In all cases, the a-carbon of the amino acid will become a transitory carbanlon (C α̅) and one of the three substituents, H, R or COOH will be pulled out in the form of cation (H⁺, R⁺ or COOH⁺ = CO₂ + H⁺).

It must be noted that pyridoxal phosphate cannot be synthesized in our organism, which explains the vitamin B₆ activity shown by pyridoxal, pyridoxine (the corresponding primary alcohol) and pyridoxamine (the corresponding primary amine which is formed intermediately during the transamination process).

A. Decarboxylation:

We have already studied the mechanism of this reaction (fig. 7-1) which leads to an amine. Some of these, amines have an important physiological or pharmacological role and are therefore sometimes called biogenic amines. The table below indicates — for some amino acids — the corresponding amine and its localization or role.

Decarboxylations are catalyzed by decarboxylases, pyridoxal phosphate-containing enzymes, present in microorganisms and animal tissues. Intestinal bacteria especially, have enzymes which can decarboxylate lysine and ornithine respectively into cadaverine and putrescine, amines which are always present in small quantities in the intestine, but can cause intoxications if their con­centration increases due to abnormal intestinal fermentations.

Bacterial decarboxylases have an extremely narrow specificity, with the result that a purified decarboxylase can permit the quantitative titration — by measuring the CO₂ liberated — of the corresponding amino acid in a complex mixture.

B. Aldolization:

By analogy with the cleavages of cis-diol bonds of carbohydrates this is an elimination of the side chain of serine and threonine transformed into glycine by cleavage of Cα — Cβ bond:

In the case of threonine (R = CH₃) the enzyme is called threonine-aldolase. It releases glycine and acetaldehyde which is transferred to the coenzyme thiamine-pyrophosphate (see oxidative decarboxylation of pyruvic acid). In the case of serine (R = H) it is serine-aldolase which, in addition to glycine, releases formaldehyde transferred to tetrahydrofolate (FH₄).

C. Transamination:

This term denotes the reversible transfer of the amino group from an amino acid to a α-keto acid; there is no liberation of NH₃ unlike during deamination which will be studied in the following.

The reactive mechanism is illustrated at the bottom of figure 7-1; in fact, there are two steps, of which one is the reverse of the other; they can be summarized as follows:

As we will note in the following, transamination is a very important reaction, for the formation of amino acids as well as the transformations they undergo. Transaminases are universally distributed: they are found in bacteria, plants and animals.

There is a large number of transaminases specific of various amino acids, but two of them are particularly abundant in animal tissues and were studied in detail; they catalyze the following 2 reactions:

L-glutamic acid + oxalo acetic acid ←→ α-ketoglutaric acid + L-aspartic acid

L-glutamic acid + pyruvic acid ←→ α-ketoglutaric acid + L-alanine

It is observed that in both cases, the amino group of glutamic acid is transferred to a α-keto acid to form the corresponding amino acid. Glutamic acid is a major participant in transamination reactions; we will see in the following that it is very easily formed by binding of NH₃ to α-ketoglutaric acid (see fig. 7-4); its amino group can then be rapidly transferred by transamination and found in the other amino acids.

It must be noted that transamination is a transfer of primary amino group (NH₂) of a donor a-amino acid to an acceptor α-keto acid. This process cannot therefore apply to proline whose nitrogen group is a secondary amine. On the contrary, transamination can take place on a primary amino group situated at the end of a side chain of amino acid. An exchange, primary amine ←→ aldehyde will then take place (see fig. 7-21).

D. Racemization:

The mechanism resembles that of transamination in the sense that proton H⁺ is pulled out from α-carbon by the effect of a proton attracting amino acid belonging to the active site of the racemase. The latter can then add another proton H⁺ through a proton donor amino acid but on the other side of the plane of the coenzyme. There is therefore inversion of the configuration of α-carbon of the substrate amino acid.

E. Reactions of α-β and α-γ Elimination:

Here again the enzymatic mechanism resembles that of transamination and also begins by the extraction of proton H⁺ from α-carbon of the substrate amino acid. These reactions are found in alcohol and sulphur-containing amino acids.

The enzyme then pulls out the alcohol or sulphur group situated on β or γ carbon, which gave the name of reactions of α-β or α- γ elimination, and produces an unsaturated, unstable intermediate susceptible of other trans­formations (spontaneous or enzymatic).

We will illustrate these reactions by two simple examples: the deamination of serine and cysteine by the action of serine-dehydratase and cysteine-desulphydrase. The action of these two en­zymes containing pyridoxal phosphate results in both cases in the release of an α-β ethylenic amino acid (see fig. 7-2) which in aqueous solution exists in tautomeric imino acid form and the latter is spontaneously hydrolyzed by water into ammonia and pyruvic acid.

Similar mechanisms and enzymes exist for threonine (dehydration) and methionine (removal of CH₃—SH group) as well as for homoserine and homocysteine. We will find them especially in connection with reactions called transulphuration reactions (see metabolism of sulphur-containing amino acids).

It is noted that in these examples the actual enzymatic reaction is an elimination of water or of a sulphur-containing compound, deamination being a non-enzymatic consequence of this elimination.

In conclusion, whatever the example, an enzyme containing pyridoxal phos­phate always acts by mechanisms of transfer of electrons and protons (plus other groups, alcohol or sulphur-containing groups in some particular cases).

2. Deamination:

A. Oxidative Deamination:

As shown by figure 7-3, this essentially irreversible process takes place in two steps. In a first phase, the enzyme catalyzes a dehydrogenation of the amino acid into an imino acid; this enzyme is a flavoprotein and the 2 H atoms removed from the substrate reduce FAD to FADH₂ (or FMN to FMNH₂).

Deamination is therefore a non-enzymatic consequence of the enzymatic process of dehydrogenation. The imino acid is hydrolyzed spontaneously into α-keto acid + ammonia. As regards the flavin coenzyme, it is reoxidized, generally by molecular oxygen; this leads to the formation of hydrogen peroxide which can then be decomposed by a catalase; but other electron acceptors can also permit the reoxidation of FADH₂.

Amino acid oxidases are stereospecific enzymes: in higher animals one has thus identified a L-amino acid oxidase (liver and kidney) not very abundant and not very active, which acts neither on glycine, nor on alcohol-containing, sulphur-containing, basic and acid amino acids.

One has also characterized a D-amino acid oxidase (liver and kidney) very abundant and very active on most D-amino acids and on glycine; this abundance is paradoxical at first sight considering the absence of corresponding substrates (D-amino acids) in higher animals.

Glutamic acid is deaminated by a particular enzyme, L-glutamate- dehydrogenase, an enzyme which contains NAD or NADP depending on the organisms, and catalyzes the reaction represented in figure 7-4.

Contrary to other oxidative deaminations, this reaction is reversible and it is particularly important as a reducing animation; this is indeed one of the main processes of fixation of ammonia in organic compounds and the amino group thus formed can easily be transferred from glutamic acid to other amino acids by trans­amination.

Moreover, this conversion from glutamic acid to α-keto glutaric acid (which is one of the compounds of the Krebs cycle) and vice-versa constitutes one of the contact points between the carbohydrate and protein metabolisms.

B. Desaturating Deamination:

It concerns only a small number of amino acids. In this process the enzyme catalyzes the removal of a molecule of ammonia with formation of a double bond between the α and β carbons. We will cite the deamination of L-aspartic acid into fumaric acid (fig. 7-5), a reversible reaction which permits the fixation of HN₃ in a compound of the Krebs cycle. This reaction is catalyzed by aspartate-ammonium lyase, an enzyme present in microorganisms and plants.

In the same manner are deaminated histidine in animals and phenylalanine in plants.