Synthesis and Absorption of Amino Acids | Biochemistry

In this article we will discuss about the synthesis and absorption of amino acids.

Synthesis of Amino Acids:

E.coli and many other micro-organisms can be grown in an inorganic medium with glucose added as the only source of carbon, which definitely shows that all amino acids can be synthesized by these cells from glucose. It may be considered that plants and other photosynthetic organisms synthesize the 20 amino acids from inorganic substances, because they form carbohydrates and thereby all organic substances by reduction of CO₂.

The synthesis of amino acids can be divided in several steps: formation of ammonia, incorporation of ammonia in an organic compound (generally in the form of glutamic acid), synthesis of the carbon skeleton of amino acids (i.e. formation of the corresponding α-keto acids) and lastly, transfer of the amino group from the glutamic acid to these various α-keto acids by transamination.

A. Formation of Ammonia:

Most microorganisms and plants use the ammonia formed during the degradation of nitrogenous organic substances.

But we must mention here the formation of ammonia from inorganic substances: atmospheric nitrogen and the nitrite and nitrate ions:

a) Fixation of Atmospheric Nitrogen in the Form of Ammonia:

This is a reduction of N₂ to 2NH₃, in other words, an addition of electrons to the two nitrogen atoms, a process whose mechanisms is not yet fully understood.

This formation of NH₃ takes place:

i. On the one hand, in microorganisms living independently: aerobic soil bacteria (of the genus Azotobacter) or anaerobic soil bacteria (of the genus Clostridium), photosynthetic bacteria, etc.

On the other hand, in some bacteria of the genus Rhizobium living in symbiosis with higher plants (for example, of the Leguminosae family) and forming on the roots of these plants, nodules where nitrogen fixation takes place.

b) Reduction of Nitrates and Nitrites to Ammonia:

Formation of Nitrites and Nitrates:

They can be formed directly from atmospheric nitrogen oxidized by lightenings during storms. But they mainly originate from the oxidation of NH₃; actually the ammonia formed either by decomposition of organic material, or by nitrogen fixation, undergoes nitrification, a process that can be summarized by the following 2 reactions:

i. In Nitrifying Bacteria of the Genus Nitrosomonas:

NH₃ + 3/2 O₂ → NO₂^– + H₂O + H⁺ (ΔG₀ = -66.5 kcal)

ii. In Nitrifying Bacteria of the Genus Nitrobacter:

NO₂^– + 1/2 O₂ → NO₃^– (ΔG₀ = -17.5 kcal)

These 2 groups of bacteria are autotrophic and synthesize all their organic compounds from CO₂ using, not light energy like the photosynthetic organisms, but the energy produced by the oxidation of inorganic compounds during the exergonic reactions written above, hence the name chemo-autotrophs given to these bacteria.

Assimilation of Nitrites and Nitrates:

This is the name given to the process used by diverse organisms — especially plants — to reduce nitrates and nitrites to NH₃. The first step is catalyzed by a nitrate-reductase, an enzyme present in various organisms (bacteria, fungi, plants) and which catalyzes the reduction of NO₃^– to NO₂^– with NADH, NADPH or FADH₂ as the source of electrons.

Then the nitrite formed is itself reduced by a nitrite-reductase and finally NH₃ is obtained, after passing through a number of intermediates (apparently including hydroxylamine), and thanks to flavoproteins containing molybdenum indispensable for their activity.

B. Incorporation of Ammonia into an Organic Compound:

The four most important processes permitting the “fixation” of NH₃ into organic compounds have already been studied; they are:

i. Reducing amination of α-ketoglutaric acid to glutamic acid by glutamate- dehydrogenase.

ii. Amination of fumaric acid to aspartic acid

iii. Formation of carbamyl-phosphate

iv. Formation of an amide like glutamine

The diverse modes of formation of ammonia and its incorporation into organic compounds can be considered within the general scope of the nitrogen cycle, which can be represented in a very simplified form (see fig. 7-6).

C. Synthesis of the Carbon Skeleton of Amino Acids:

The α-keto acids corresponding to a number of amino acids are common products of the intermediate metabolism, especially of glycolysis and Krebs cycle; this is particularly the case of pyruvic acid (α-keto acid corresponding to alanine), α-keto glutaric acid (corresponding to glutamic acid), oxaloacetic acid (corresponding to aspartic acid).

Some other α-keto acids derive more or less directly from compounds of the intermediate metabolism, for example, 3-phospho-hydroxy-pyruvic acid (corresponding to phospho-serine) which results from 3-phospho-giyceric acid by dehydrogenation (see fig. 7-8).

But while microorganisms and plants are capable of forming all the α-keto acids, higher animals and particularly man, are incapable of achieving the synthesis of some of them; these α-keto acids or the corresponding amino acids must be supplied to them through diet; we will study in the following these amino acids called “essential”.

As regards the organisms capable of synthesizing all the α-keto acids, the reactions required are sometimes numerous and complex — especially for the aromatic amino acids — and the biosynthesis of all amino acids.

D. Transfer of the Amino Group to the α-keto Acids:

Generally, amino acids are formed form the corresponding α-keto acids, by transamination. Since glutamic acid is the principal compound resulting from the incorporation of NH₃ in organic form, it will be the main donor of the amino group in transamination reactions. As for α-keto acids, they-are produced by the pathways of carbohydrate catabolism (pyruvic acid, oxaloacetic acid) or derive from these pathways.

The formation of various amino acids from ammonia and α-keto acids can be summarized in a simple diagram (see fig. 7-7):

However, it must be noted that of the 20 amino acids which constitute the proteins, 9 are not synthesized according to this general procedure; Asn, Gin, Thr, Pro, Cys, Met, Trp, Lys and Arg. The corresponding α-keto acids are indeed not produced by one of the pathways of carbohydrate catabolism (glycolysis, Krebs cycle or cycle of pentoses).

These amino acids are therefore formed from the others by special reactions of radical transformation called conversion reactions. On the other hand, the synthesis of some amino acids requires both a radical conversion and a transamination reaction (valine, isoleucine, leucine).

Lastly, depending on the organism considered, glycine can be synthesized either by conversion or by transamination. In the following sections we will see how glycine is synthesized from serine (see fig. 7-9), cysteine from methionine, proline from glutamic acid, lysine, threonine and isoleucine from aspartic acid.

Absorption of Preformed Amino Acids:

A. Essential Amino Acids:

In mammals — and especially in man — a number of amino acids cannot be formed by the mechanisms we have just described (see fig. 7-7), either because the corresponding α-keto acids are not present (and cannot be synthesized), or because they can undergo neither amination nor transamination.

In man, these amino acids are 8 in number: three amino acids with hydrocarbon side chain (Leu, Ile, Val), one basic amino acid (Lys), one hydroxylated amino acid (Thr), one sulphur-containing amino acid (Met) and 2 aromatic amino acids (Phe and Trp). They are called “indispensable” or “essential” amino acids and must be supplied by diet.

There are 9 amino acids essential for the growth of rat — all the 8 already cited for man and additionally, histidine — as may be proved by dietary experiments in which the animal is fed, not with proteins but with an artificial mixture of free amino acids, omitting by turn each amino acid: if the amino acid omitted is essential, the biosynthesis of proteins is perturbed.

Theoretically, normal life and growth can be ensured by providing the essential amino acids and ammonium salts to allow the formation of other amino acids.

But in reality, animal as well as human diet includes proteins which yield the essential amino acids as well as those which could be synthesized: one can thus avoid consuming the corresponding precursors like the α-keto acids participat­ing in the glycolysis or Krebs cycle (they can thus continue to function in the oxidation of carbohydrates or fatty acids which produces ATP).

B. Hydrolysis of Proteins:

The dietary proteins must hydrolyzed to amino acids in the digestive tract. As already mentioned in the case of polysaccharides, in general, macro- molecules are not properly absorbed.

Moreover, in the case of proteins, an absorption without previous hydrolysis would be of no interest because the proteins are specific: we do not need dietary plant or animal proteins; we need proteins of our own with very different sequences, resulting from the expres­sion of our genes.

Lastly, the introduction of foreign proteins in our organism may be fraught with real dangers and can be followed by reactions ranging from relatively minor allergic manifestations (skin reactions in some persons follow­ing ingestion of fish, crustacea, or hay fever in presence of some pollens), to serious conditions of anaphylactic shock (observed for example during serotherapy when the patient is injected with rather large and repeated quan­tities of horse serum containing serum proteins of this animal and especially the antibody desired).

For all these reasons the proteins must be hydrolyzed to amino acids and the latter can then be absorbed, not by simple diffusion, but thanks to specific proteins called permeases.

In proteins, amino acids are linked to one another by the peptide linkage and one might think that a single enzyme would suffice for the complete hydrolysis of all the proteins as there is only one type of bond to be split.

In reality, the proteolytic enzymes, or proteases, or peptidases, reveal a more or less pronounced specificity vis-a-vis the position of the peptide bond in the chain and the nature of the amino acids involved in this linkage. As a result, the rapid and complete hydrolysis of a protein requires the participation of several enzymes capable of attacking the polypeptide chain at diverse points.

a) Endopeptidases:

They are the enzymes which catalyze the hydrolysis of peptide bonds within the chains.

There are 3 important endopeptidases in our digestive tract:

i. Pepsin is secreted by the gastric mucosa in the form of inactive pep­sinogen, which is transformed — by removal of a number of small peptides — into an active enzyme whose optimum pH is very low (around 2). As men­tioned in fig. 2-26, pepsin preferentially hydrolyzes the peptide bonds involving the amino group of an aromatic amino acid (Phe, Tyr);

ii. Trypsin is contained in the pancreatic juice and continues in the intestine the digestion of proteins started in the stomach by pepsin. It is secreted by the pancreas, also in the form of an inactive precursor — trypsinogen — whose transformation into trypsin is an activation which is either autocatalytic (under the influence of trypsin itself), or catalyzed by another proteolytic enzyme such as enterokinase secreted by the duodenal mucosa, and which consists in the removal of a hexapeptide. Trypsin has an optimum pH around 8; it preferen­tially hydrolyzes the peptide bonds in which a basic amino acid (Arg, Lys) participates by its carboxyl (see fig. 2-26).

iii. Chymotrypsin is also secreted by the pancreas in the form of a zymogen, the chymotrypsinogen, transformed into chymotrypsin by the action of trypsin. It also has an optimum pH near 8 and preferentially splits the peptide bonds in which aromatic amino acids (Trp, Tyr, Phe) are involved; but contrary to pepsin which splits the bonds in which amino groups of aromatic acids are involved, chymotrypsin splits those in which carboxylic groups are involved (see fig. 2-26).

The action of these 3 endopeptidases on the dietary proteins leads to a mixture of peptides of varied sizes and some free amino acids. These peptides will then be attacked by exopeptidases.

b) Exopeptidases:

They are the enzymes which catalyzes the hydrolysis of peptide bonds at the end of chains:

i. The pancreatic carboxypeptidases hydrolyze the peptide bond in which the C-terminal amino acid is involved;

ii. The intestinal aminopeptidases hydrolyze the peptide bond in which the N-terminal amino acid is involved. As indicated by its name, the leucine- aminopeptidases has a preference for leucine, but this is not an absolute specificity and how this enzyme, as also the carboxypeptidases, permit the determination of the nature of amino acids situated at the ends of the peptide chains;

iii. The intestinal dipeptidases can split the dipepttdes; but a particular en­zyme, the prolidase, is required for hydrolyzing the dipeptides in which proline is the amino acid having the free carboxyl because this is not a conventional peptide bond.

We only mentioned the proteolytic enzymes enabling the hydrolysis of dietary proteins in our digestive tract. But many animal cells also contain endopeptidases called cathepsins A, B and C whose specificity is analogous respectively to that of pepsin, trypsin and chymotrypsin; these cathepsins are responsible for the post-mortem autolysis but their role in living cells is not yet fully understood. Lastly, it must be said that there are other proteolytic en­zymes in living organisms: subtilisin (in Bacillus subtilis), papain (in the papaya tree, a higher plant), etc.