ГЛАВА КНИГИ

ГЛАВА КНИГИ

Aromatic amino acids

Phenylalanine, tyrosine, and tryptophan have aromatic side chains

The nonpolar aliphatic and aromatic amino acids are normally buried in the protein core and are involved in hydrophobic interactions with one another. Tyrosine has a weakly acidic hydroxyl group and may be located on the surface of proteins. Reversible phosphorylation of the hydroxyl group of tyrosine in some enzymes is important in the regulation of metabolic pathways. The aromatic amino acids are responsible for the ultraviolet absorption of most proteins, which have absorption maxima ~280 nm. Tryptophan has a greater absorption in this region than phenylalanine or tyrosine. The molar absorption coefficient of a protein is useful in determining the concentration of a protein in solution, based on spectrophotometry. Typical absorption spectra of aromatic amino acids and a protein are shown in Fig. 2.3 .

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Fig. 2.3

Ultraviolet absorption spectra of the aromatic amino acids and bovine serum albumin.

(A) Aromatic amino acids such as tryptophan, tyrosine, and phenylalanine have absorbance maxima at 260–280 nm. Each purified protein has a distinct molecular absorption coefficient at around 280 nm, depending on its content of aromatic amino acids. (B) A bovine serum albumin solution (1 mg dissolved in 1 mL of water) has an absorbance of 0.67 at 280 nm using a 1-cm cuvette. The absorption coefficient of proteins is often expressed as E _1%(10 mg/mL solution). For albumin, E _1%280 _nm= 6.7. Although proteins vary in their Trp, Tyr, and Phe content, measurements of absorbance at 280 nm are useful for estimating protein concentration in solutions.

Neutral polar amino acids

Neutral polar amino acids contain hydroxyl or amide side chain groups. Serine and threonine contain hydroxyl groups. These amino acids are sometimes found at the active sites of catalytic proteins, enzymes ( Chapter 6 ). Reversible phosphorylation of peripheral serine and threonine residues of enzymes is also involved in regulation of energy metabolism and fuel storage in the body ( Chapter 12 ). Asparagine and glutamine have amide-bearing side chains. These are polar but uncharged under physiologic conditions. Serine, threonine, and asparagine are the primary sites of linkage of sugars to proteins, forming glycoproteins ( Chapter 17 ).

Acidic amino acids

Aspartic and glutamic acids contain carboxylic acids on their side chains and are ionized at pH 7 and, as a result, carry negative charges on their β- and γ-carboxyl groups, respectively. In the ionized state, these amino acids are referred to as aspartate and glutamate, respectively.

Basic amino acids

The side chains of lysine and arginine are fully protonated at neutral pH and, therefore, positively charged. Lysine contains a primary amino group (NH ₂ ) attached to the terminal ε -carbon of the side chain. The ε -amino group of lysine has a p K _a ≈ 11. Arginine is the most basic amino acid (p K _a ≈ 13), and itsguanidine group exists as a protonated guanidinium ion at pH 7.

Histidine (p K _a ≈ 6) has an imidazole ring as the side chain and functions as a general acid–base catalyst in many enzymes. The protonated form of imidazole is called an imidazolium ion.

Sulfur-containing amino acids

Cysteine and its oxidized form, cystine, are sulfur-containing amino acids characterized by low polarity. Cysteine plays an important role in the stabilization of protein structure because it can participate in the formation of a disulfide bond with other cysteine residues to form cystine residues, crosslinking protein chains and stabilizing protein structure. Two regions of a single polypeptide chain, remote from each other in the sequence, may be covalently linked through a disulfide bond (intrachain disulfide bond). Disulfide bonds are also formed between two polypeptide chains (interchain disulfide bond), forming covalent protein dimers. These bonds can be reduced by enzymes or reducing agents, such as 2-mercaptoethanol or dithiothreitol, to form cysteine residues. Methionine is the third sulfur-containing amino acid and contains a nonpolar methyl thioether group in its side chain.

Proline, a cyclic imino acid

Proline is different from other amino acids in that its side chain pyrrolidine ring includes both the α-amino group and the α-carbon. This imino acid forces a “bend” in a polypeptide chain, sometimes causing abrupt changes in the direction of the chain.

Classification of amino acids based on the polarity of the amino acid side chains

Table 2.2 depicts the functional groups of amino acids and their polarity (hydrophilicity). Polar side chains can be involved in hydrogen bonding to water and to other polar groups and are usually located on the surface of the protein. Hydrophobic side chains contribute to protein folding by hydrophobic interactions and are located primarily in the core of the protein or on surfaces involved in interactions with other proteins.

Table 2.2

Summary of the functional groups of amino acids and their polarity

Amino acids	Functional group	Hydrophilic (polar) or hydrophobic (nonpolar)	Examples
acidic	carboxyl, –COOH	polar	Asp, Glu
basic	amine, –NH₂	polar	Lys
	imidazole	polar	His
	guanidino	polar	Arg
neutral	glycine, –H	nonpolar	Gly
	amides, –CONH ₂	polar	Asn, Gln
	hydroxyl, –OH	polar	Ser, Thr,
	sulfhydryl, –SH	nonpolar	Cys
aliphatic	hydrocarbon	nonpolar	Ala, Val, Leu, Ile, Met, Pro
aromatic	C-rings	nonpolar	Phe, Trp, Tyr

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Ionization state of an amino acid

Amino acids are amphoteric molecules - they have both basic and acidic groups

Monoamino and monocarboxylic acids are ionized in different ways in solution, depending on the solution's pH. At pH 7, the “zwitterion” ⁺ H ₃ N–CH ₂ –COO ⁻is the dominant species of glycine in solution, and the overall molecule is therefore electrically neutral. On titration to acidic pH, the α-amino and carboxyl groups are protonated, yielding the cation ⁺ H ₃ N–CH ₂ –COOH, whereas titration with alkali yields the anionic H ₂ N–CH ₂ –COO ⁻ species:

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p K _a values for the α-amino and α-carboxyl groups and side chains of acidic and basic amino acids are shown in Table 2.3 . The overall charge on a protein depends on the contribution from basic (positive charge) and acidic (negative charge) amino acids, but the actual charge on the protein varies with the pH of the solution. To understand how the side chains affect the charge on proteins, it is worth recalling the Henderson–Hasselbalch (H-H) equation.

Table 2.3

p K _avalues for ionizable groups in proteins

Group	Acid (protonated form) (conjugate acid)	H ⁺+ base (unprotonated form) (conjugate base)	p K_a
terminal carboxyl residue (α-carboxyl)	–COOH (carboxylic acid)	–COO ⁻+ H ⁺(carboxylate)	3.0–5.5
aspartic acid (β-carboxyl)	–COOH	–COO ⁻+ H ⁺	3.9
glutamic acid (γ-carboxyl)	–COOH	–COO ⁻+ H ⁺	4.3
histidine (imidazole)	Открыть изображение в полном размере	Открыть изображение в полном размере	6.0
terminal amino (α-amino)	–NH ₃⁺(ammonium)	–NH ⁺+ H ⁺(amine)	8.0
cysteine (sulfhydryl)	–SH (thiol)	–S ⁻+ H ⁺(thiolate)	8.3
tyrosine (phenolic hydroxyl)	Открыть изображение в полном размере	Открыть изображение в полном размере	10.1
lysine (ε-amino)	–NH ₃⁺	–NH ₂+ H ⁺	10.5
arginine (guanidino)	Открыть изображение в полном размере	Открыть изображение в полном размере	12.5

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Actual p K _avalues may vary by several pH units, depending on temperature, buffer, ligand binding, and especially neighboring functional groups in the protein.

Henderson–Hasselbalch equation and p K _a

The H-H equation describes the titration of an amino acid and can be used to predict the net charge and isoelectric point of a protein

The general dissociation of a weak acid, such as a carboxylic acid, is given by the following equation:

HA⇄H++A−HA⇄H++A−

where HA is the protonated form (conjugate acid, or associated form), and A ⁻ is the unprotonated form (conjugate base, or dissociated form).

The dissociation constant ( K _a ) of a weak acid is defined as the equilibrium constant for the dissociation reaction (1) of the acid:

Ka=[H+][A−][HA]Ka=[H+][A−][HA]

The hydrogen ion concentration [H ⁺ ] of a solution of a weak acid can then be calculated as follows. Eq. (2) can be rearranged to give

[H+]=Ka×[HA][A−][H+]=Ka×[HA][A−]

Eq. (3) can be expressed in terms of a negative logarithm:

–log[H+]=−logKa–log[HA][A−]–log[H+]=−logKa–log[HA][A−]

Because pH is the negative logarithm of [H ⁺ ] (i.e., −log[H ⁺ ]), and p K _a equals the negative logarithm of the dissociation constant for a weak acid (i.e., −log K _a ), the Henderson–Hasselbalch Eq. (5) can be developed and used for analysis of acid–base equilibrium systems:

pH=pKa+log[A−][HA]pH=pKa+log[A−][HA]

For a weak base, such as an amine, the dissociation reaction can be written as

RNH3+⇄H++RNH2RNH3+⇄H++RNH2

and the Henderson–Hasselbalch equation becomes

pH=pKa+log[RNH2][RNH3+]pH=pKa+log[RNH2][RNH3+]

From Eqs. (5) and (7) , it is apparent that the extent of protonation of acidic and basic functional groups, and therefore the net charge of an amino acid, will vary with the p K _a of the functional group and the pH of the solution. For alanine, which has two functional groups with p K _a = 2.4 and 9.8, respectively ( Fig. 2.4 ), the net charge varies with pH, from +1 to −1. At a point intermediate between p K_a1 and p K _a2 , alanine has a net zero charge. This pH is called its isoelectric point, pI ( Fig. 2.4 ).

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Fig. 2.4

Titration of amino acid.

The curve shows the number of equivalents of NaOH consumed by alanine while titrating the solution from pH 0 to pH 12. Alanine contains two ionizable groups: an α-carboxyl group and an α-amino group. As NaOH is added, these two groups are titrated. The p K _aof the α-COOH group is 2.4, whereas that of the α-NH ₃⁺group is 9.8. At very low pH, the predominant ion species of alanine is the fully protonated, cationic form:

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At the mid-point in the first stage of the titration (pH 2.4), equimolar concentrations of proton donor and proton acceptor species are present, providing good buffering power.

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At the mid-point in the overall titration (pH 6.1) the zwitterion is the predominant form of the amino acid in solution. The amino acid has a net zero charge at this pH - the negative charge of the carboxylate ion being neutralized by the positive charge of the ammonium group.

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The second stage of the titration corresponds to the removal of a proton from the –NH ₃⁺group of alanine. The pH at the mid-point of this stage is 9.8, equal to the p K _afor the –NH ₃⁺group. The titration is complete at a pH of about 12, at which point the predominant form of alanine is the unprotonated, anionic form:

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The pH at which a molecule has no net charge is known as its isoelectric point, pI. For alanine, it is calculated as follows:

pI=pKa1+pKa22=(2.4+9.8)2=6.1pI=pKa1+pKa22=(2.4+9.8)2=6.1

Stereochemistry: Configuration at the α-carbon and d- and l-isomers