Structural Biochemistry - Study Guide - E. A. Bessolitsyna 2015

Amino Acids contain an amino group and a carboxyl group as functional groups (Figure 57). In α-amino acids, both are attached to the same (α) carbon atom. There are about 300 amino acids in nature, yet only 20 of them are found in Proteins. Complete Hydrolysis of Proteins yields 20 L-α-amino acids. The exact same 20 Amino acids are present in protein molecules across all forms of life—plants, animals, and microorganisms. However, certain proteins contain derivatives of specific amino acids that are formed after the standard Amino acids have already been incorporated into the protein molecule. With the exception of Glycine, where R is a hydrogen atom, all amino acids have four different groups attached to the α-carbon atom. Due to the tetrahedral arrangement of these four distinct groups around the α-carbon atom, amino acids exhibit optical activity (The ability to rotate the plane of polarization of plane-polarized light). Some amino acids that make up proteins are dextrorotatory at pH 7.0, while others are levorotatory; nevertheless, all of them share the absolute configuration of L-glyceraldehyde and are therefore classified as L-α-amino acids. Threonine and isoleucine each contain 2 chiral centers, meaning there are 4 stereoisomers for each of these amino acids. Proteins contain only one of them. Optical stereoisomerism is of paramount importance for the realization of biochemical mechanisms.

L-α-Amino acids are the monomers from which Peptides and Proteins are constructed. Only twenty amino acids—the so-called Proteinogenic Amino Acids—are incorporated into the polypeptide chain during Translation. Subsequently, As a result of post-translational modification reactions, some of them are converted into other amino acid residues (for example, Cysteine into cystine, Serine into phosphoserine, etc.). A much greater number of amino acids exist in nature. For instance, numerous "non-proteinogenic" amino acids are found in peptide Antibiotics, the synthesis of which proceeds via a non-ribosomal mechanism, and a whole range of "non-proteinogenic" amino acids participate in metabolic processes. These include, in particular, homoserine, argininosuccinic acid, and aspartic acid semialdehyde—intermediates in The Biosynthesis of proteinogenic amino acids; azetidinecarboxylic acid, a common component of plant Cell sap; taurine, which is found in Bile as part of bile acid conjugates; and γ-aminobutyric acid (GABA), a neurotransmitter. Minor amino acids such as 4-hydroxyproline and 5-hydroxylysine are found in the fibrillar Connective Tissue protein Collagen, while N-methyllysine is found in the Muscle protein Myosin. Phosphorylation of the hydroxyl groups of serine, threonine, and Tyrosine residues is also widespread. Desmosine has a much more complex Structure, being a component of the protein Elastin. Desmosine is formed by 4 molecules of the principal amino acid Lysine, linked together via their R-groups to create a substituted pyridine ring (effectively four lysine molecules radiating from a single center). This results in The formation of cross-links between The polypeptide chains of the protein. A distinctive feature of desmosine is its ability to stretch in two directions, which contributes to the elasticity of connective tissue, and so on.

Proteinogenic amino acids are incorporated into proteins on the ribosome; 20 such amino acids have been identified (21 if selenocysteine is included).

Unlike proteinogenic amino acids, non-proteinogenic amino acids are not incorporated on The Ribosome and are not encoded in The Genome. Non-proteinogenic amino acids perform a multitude of other Functions. While they may not be constituents of proteins synthesized via ribosomal template mechanisms, they can still be found in Polypeptides as a result of post-synthetic modifications.

Figure 57. General formula of proteinogenic amino acids

All proteinogenic amino acids are L-α-amino acids.

Proteinogenic amino acids are classified according to The properties of their side chains: amino acid residues with hydrophobic chains, hydrophilic neutral amino acids, amino acids containing aromatic radicals, basic side chains, and acidic side chains (Figure 58).

Figure 58. Structural formulas of proteinogenic amino acids. Proline is an imino acid; aspartic and glutamic acids are acidic amino acids; lysine, Arginine, and Histidine are basic amino acids.

Amino acid residues with hydrophobic chains of varying geometry make it possible to form a compact interior core that stabilizes Protein Structure and establishes hydrophobic contacts with its ligands. Glycine, which lacks a side chain, is indispensable for allowing peptide chains to approach closely and facilitates their conformational turns.

Hydrophilic neutral amino acids—serine, threonine, asparagine, and glutamine—participate in the formation of hydrogen bonding networks and ensure protein Hydration. Side chains containing ionogenic groups (residues of glutamic and aspartic acids, histidine, lysine, and arginine), In addition to forming Hydrogen Bonds, participate in ionic interactions both within the protein and with other molecules. Cysteine residues allow proteins to participate in oxidation-reduction processes and also serve as precursors to cystine residues by forming disulfide bridges, which act as additional stabilizing factors for protein structure.

Figure 59. Structure of non-proteinogenic amino acids. A — hydroxylysine, B — methyllysine, C — hydroxyproline, D — desmosine.

At the same time, It is worth noting that the variety of functional groups in proteinogenic amino acids is not all that vast. However, their chemical capabilities are drastically enhanced within the framework of specific protein structures through the formation of spatially organized ensembles. Nevertheless, for many tasks, the protein's functional groups alone are not sufficient; to solve them, specifically bound ligands are recruited—such as Metal Ions, chromophore groups, Coenzymes, and the like.

The solubility of an amino acid is determined by its side chain, the Hydrophobicity of which decreases The amino acid's solubility. None of the primary amino acids absorb light in the visible region, and only three absorb light in the near ultraviolet. Tryptophan absorbs the most strongly (with an absorption maximum around 280 nm), tyrosine significantly more weakly (280 nm), and phenylalanine even more weakly (around 260 nm). Most proteins contain tyrosine, which is why measuring optical density at 280 nm is a common METHOD FOR DETERMINING protein concentration. Cystine exhibits weak absorption at 240 nm due to its disulfide group. All amino acids absorb in the far ultraviolet (<220 nm) (Figure 60).

Figure 60. Absorption spectra of amino acids

Acid-Base Properties

The acid-base properties of amino acids are determined by the simultaneous presence of amino and carboxyl groups in their structure, which, being extremely close to one another, exert a profound mutual influence. In a neutral medium, over a fairly wide pH range, α-amino acids exist as dipolar ions; therefore, representing the formula of amino acids in the conventional manner—for example, glycine as —NH₂—CH₂—COOH—is merely a convention. For neutral solutions, it would be more accurate to write NH₃⁺—CH₂—COO^-.

It is precisely in this form of dipolar ions—zwitterions (an obsolete term is "betaine-like structure")—that amino acids exist in neutral solutions and crystals. The electron-withdrawing Influence of the positively charged ammonium group in the α-position makes the α-carboxyl group significantly stronger compared to the carboxyl groups of aliphatic acids. Thus, while the pK_a of acetic acid (the pH at which dissociation is 50%) is 4.7, the carboxyl pK_a of glycine is 2.34. Approximately similar pK_a values are characteristic of the carboxyl groups of other α-amino acids. Naturally, carboxyl groups located at a distance from the α-ammonium group—such as the carboxyls of aspartic or glutamic acids—do not experience such a significant positive charge effect and, in their acidic properties and ability to donate and accept protons, approach ordinary Fatty acids. The thiol (or sulfhydryl) group of cysteine and the hydroxyl group of tyrosine possess very weak acidic properties. At pH = 7, the SH group is ionized by approximately 8%, and the OH group by 0.01%.

Similarly, the α-amino group in α-amino acids is influenced by the carboxylate anion attached to the same carbon atom, making it considerably less basic than the amino group of primary aliphatic amines—its pK_a is 9.6 – 9.7, in contrast to 10.7 for ethylamine. METABOLISM/18.html">The Influence of the carboxyl group on the Properties of the amino group, particularly on its basicity, decreases as the distance between them increases; therefore, the ε-amino group of lysine is virtually indistinguishable in its chemical characteristics from aliphatic amines, with a pK_a of 10.5.

Figure 61. Change in amino acid charge as a function of pH

If an acid is added to a neutral solution of an α-amino acid containing dipolar ions, thereby increasing the concentration of hydrogen ions, the latter will bind to the carboxylate groups, converting them into uncharged carboxyl groups. In this process, the amino acid—whose charges were initially balanced—will acquire a net positive charge and, specifically, will migrate toward the cathode in an electric field.

Conversely, if the hydrogen ion concentration is lowered by adding an alkali, hydroxyl groups will react with α-amino groups, releasing a proton and converting them into uncharged amino groups. As a result, the amino acid acquires a net negative charge and migrates toward the anode in an electric field. The pH at which both the amino and carboxyl groups of the amino acid are fully ionized and their charges are balanced is called the isoelectric point (p_i). For α-amino acids lacking ionizable groups in their side chain, the p_i value equals the half-sum of the pK_a values of the α-amino and α-carboxyl groups. If the amino acid contains additional ionizable groups, their contribution must be factored into the p_i calculation (Figure 61).

Chemical Reactions of Amino Acids

These reactions are classified into those characteristic of α-amino groups, α-carboxyl groups, reactions involving both groups simultaneously, and reactions involving functional groups within the side chain (radical).

Figure 62. Chemical reactions of amino acids. A—formol titration, B—acylation with acid derivatives

Reactions of Amino Groups

Overall, these reactions are analogous to those of aliphatic amines.

1. Formol titration. Formaldehyde, taken in excess, readily combines with free (unprotonated) amino groups to form methylol derivatives. As a result, the amino acid at its isoelectric point releases a proton from the zwitterion's amino group. The liberated protons can be titrated with NaOH until a phenolphthalein color change appears (pH 8.0). Formol titration is an analytical method for determining amino acid content (Figure 62, A).

2. Acylation with activated acid derivatives proceeds quite easily in the presence of a base that binds the released acid. Examples include the Acylation of Amino acids with acetic anhydride (Figure 62, B).

3. Dansylation—a reaction with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride)—allows The conversion of an amino acid into a fluorescent compound (Figure 63, A).

Figure 63. Chemical reactions of amino acids. A—dansylation, B—arylation, C—reaction with aldehydes

4. Arylation proceeds similarly using the highly reactive 1-fluoro-2,4-dinitrobenzene—a dinitrophenylation reaction that played a major role in determining peptide structures (Figure 63, B).

5. The reaction of the amino group with aldehydes leads to the formation of an unsaturated compound known as a Schiff base, in which the basicity of the nitrogen is significantly reduced (Figure 63, C). This reaction is reversible, and in an acidic environment, the aldehyde is cleaved to regenerate the amino acid. The Schiff base is characterized by the proximity of the double bond to the asymmetric center, which can promote Amino Acid Racemization due to the migration of the double bond to the carbon atom. Hydrogenation of the double bond in the Schiff base, for example with sodium borohydride, stabilizes the C–N bond. Schiff base formation is not a qualitative test for identifying amino acids, but this reaction frequently occurs within Cells during Amino acid metabolism.

6. An analytically important reaction involving carbonyl compounds is the interaction of amino acids with triketohydrindene hydrate (ninhydrin) (Figure 64, A). This reaction is the basis for both qualitative and Quantitative determination of amino acids (including automated analyzers).

Figure 64. Chemical reactions of amino acids. A—reaction with ninhydrin, B—reaction with phthalaldehyde

7. Fluorescent derivatives are formed when α-amino acids react with o-phthalaldehyde in the presence of mercaptoethanol. This reaction makes it possible to detect extremely small, picomole-range quantities of amino acids (Figure 64, B).

Reactions of Carboxyl Groups

These amino acid reactions largely correspond to those of aliphatic carboxylic acids. Esterification takes place in anhydrous alcohols in the presence of acid catalysts. For instance, phenylalanine is esterified almost quantitatively in anhydrous methanol using Hydrochloric acid or (preferably) thionyl chloride. Amino acid esters—important starting Materials for Peptide Synthesis—can undergo hydrolysis, particularly in alkaline media (saponification). Treating amino acid esters with anhydrous ammonia converts them into amides. For the temporary protection of α-carboxyl groups required in peptide synthesis, they are converted into tert-butyl esters via reaction with isobutylene in the presence of sulfuric acid. Such esters are stable in mildly alkaline environments but are selectively cleaved by acids to regenerate the free carboxyl group.

Reactions Involving Both α-Amino and Carboxyl Groups

1. Amino acid salts with certain metal ions—specifically divalent copper, nickel, and cobalt ions—are formed with the participation of both the carboxylate ion (ionic bond) and the amino group (coordination bond). This produces highly stable bicyclic (claw-like, or chelate) structures in which a metal ion is coordinated with two amino acid molecules, such as the copper salt of lysine, whose formation involves the carboxyl and amino groups at the carbon atom (Figure 65). The ε-amino group is not part of the complex and remains free, which can be utilized to carry out reactions specifically targeting only this remote amino group. Such complexes are intensely colored; for instance, copper complexes with amino acids exhibit a violet-blue color. This reaction is known as the biuret test.

Figure 65. Formation of a chelate complex

2. Reactions are known that convert amino acids into cyclic structures involving both the amino and carboxyl groups. For example, phosgene converts α-amino acids into so-called N-carboxyanhydrides, which readily polymerize with the elimination of CO₂ to form polyamino acids.

3. The reaction of amino acids with phenyl isothiocyanate is of paramount importance; it initially yields an α-amino group derivative, a phenylthiocarbamyl amino acid (Figure 66). Phenylthiocarbamyl Amino Acids and some of their analogs absorb intensely in the ultraviolet region, which is exploited in the quantitative Determination of Amino acids by High-Performance Liquid Chromatography. Acid Treatment leads to the intramolecular cyclization of the phenylthiocarbamyl amino acid, yielding a phenylthiohydantoin. These Amino Acid Derivatives play a crucial role in protein Primary Structure analysis.

Figure 66. Reaction of amino acids with phenyl isothiocyanate

4. Two molecules of the same or different Amino acids can covalently bind to each other via a substituted amide bond, termed a peptide bond, to form a dipeptide. The resulting dipeptide still retains one free (and therefore reactive) amino group and one free carboxyl group. Consequently, Other Amino Acids can sequentially attach to the dipeptide through peptide bond formation, yielding tri-, tetra-, … oligo-, and polypeptides. In an aqueous environment, the equilibrium of this reaction lies toward the formation of free amino acids. Synthesis both in vivo and in vitro proceeds indirectly following the activation of the interacting groups (most commonly the carboxyl group). For example, amino acids can first be converted into acyl chlorides, from which peptides are then synthesized. It was precisely this method that E. Fischer used to prepare peptides up to 20 amino acids long. These products exhibited properties common to proteins and their hydrolytic products, thereby proving the validity of the peptide theory of protein structure. Today, Methods have been developed for synthesizing polypeptides of arbitrary length and composition.

Reactions of Functional Groups within Side Chains (Radicals)

1. Folin's test for Sulfur-Containing Amino Acids. Upon prolonged heating, a solution containing sulfur-containing amino acids (cysteine, cystine, Methionine) and proteins comprising these amino acids turns brown, and a black precipitate of lead sulfide forms (Figure 67, A, B).

Figure 67. Chemical reactions of amino acids. A, B—Folin's test, C—xanthoproteic test

The reaction proceeds in two stages. Stage 1 involves the Cleavage of amino acid SH-groups and the conversion of sulfur from an organic compound into an inorganic one. Stage 2 involves the qualitative detection of sulfur ions in the solution. Among the functional groups found in amino acid side chains, the highly reactive sulfhydryl group of cysteine is particularly noteworthy. It easily oxidizes to form a disulfide (cystine). Treatment with trace amounts of heavy metals yields mercaptides. Since the SH-group is part of the active sites of many Enzymes, exposure to heavy metals (e.g., Ag, Hg) leads to Enzyme inactivation.

2. Xanthoproteic test. If cyclic amino acids or proteins containing them are present in the solution, a yellow coloration appears due to the nitration of the benzene ring (Figure 67, C).

Functions of proteinogenic amino acids:

They are constituents of proteins, as proteinogenic amino acids are incorporated into the polypeptide chain at the ribosome. They are encoded in the genome.

They are components of peptide antibiotics and Hormones.

They participate in metabolic processes.

They are components of peptide antibiotics and hormones.

Functions of non-proteinogenic amino acids:

A number of non-proteinogenic amino acids participate in metabolic pathways. Examples include homoserine, argininosuccinic acid, and aspartic semialdehyde, which are intermediates in the biosynthesis of proteinogenic amino acids; azetidine-2-carboxylic acid, a common component of plant cell sap; and taurine, found in bile as a component of bile acid conjugates.

γ-aminobutyric acid (GABA) acts as a neurotransmitter.

Numerous modified amino acids are incorporated into proteins, altering their properties.

Non-proteinogenic amino acids are components of antibiotics, toxins, and murein, which forms The Cell walls of Bacteria.