4.2. Properties of Common Amino Acids

Amino acids participate in a large variety of both structural and catalytic roles in biological systems. As a result, their general properties also vary.

All amino acids contain at least one acid (the carboxylic acid) and at least one base (the amine). Most amino acids broadly undergo the same chemical reactions at roughly the same rates at these positions. However, in general these two functional groups are occupied in chemical bonds in biological systems and tend not to be as relevant to discussion of their bioorganic properties.

Bioorganic chemists (and biochemists) prefer to subdivide amino acids into categories based on similar chemical properties. This helps to explain why certain amino acids fulfill certain roles in biology. There are several different ways of doing this. By far the most common is to group amino acids by the different properties given by their side chains.

4.2.1. Classification and Properties by Side Chain

The side chains (R, Figure 4.8) of amino acids vary substantially. This text will focus on the similarities, differences, and groupings for the twenty common amino acids.

Figure 4.8 – Generalized α-Amino Acid Highlighting the Side Chain.

There are several different ways of grouping these amino acids by their side chains. Some sources may describe categories differently, use different criteria, or place amino acids in different categories. At an introductory level the goal is to recognize patterns and similarities, and to be aware of what reactions and interactions the side chains may (or may not) be capable of participating in.

4.2.1.1. Hydrogen (Glycine)

The simplest of the common amino acids is glycine (Figure 4.9). Glycine’s side chain is a hydrogen. This is, debatably, an aliphatic group and glycine often behaves similarly to amino acids with aliphatic side chains (see Section 4.2.1.2). It is also semi-commonly found in similar places in proteins/enzymes.

Figure 4.9 – Common Amino Acid with Hydrogen Side Chain: Glycine.

Glycine is the only common amino acid that lacks a stereocentre. While this has some impact on its biochemical properties, the larger impact is on its size. The lack of steric bulk from the side chain means that glycine is often incorporated into places that other amino acids would be too “bulky” for. It also makes the amino acid slightly more flexible, which leads to it commonly being incorporated in areas that take advantage of this (see Section 4.5.2.4).

4.2.1.2. Aliphatic (Alanine, Valine, Isoleucine, Leucine, Proline)

Several of the common amino acids have alkyl groups as side chains (Figure 4.10). Most sources instead use the term “aliphatic” for these side chains. Aliphatic is a classical term that means “hydrocarbons that are not aromatic” (e.g. alkyl groups).

Figure 4.10 – Common Amino Acids with Aliphatic Side Chains: L-Alanine, L-Valine, L-Isoleucine, L-Leucine, and L-Proline.

These amino acids have side chains that are non-polar alkyl groups. They prefer dispersion interactions (sometimes called London forces) over all others. Recall that dispersion interactions are a type of intermolecular force where temporarily induced dipoles are slightly attracted to each other (Figure 4.11).

Figure 4.11 – Representation of Temporarily Induced Dipoles for Dispersion Interactions in Methane.

Dispersion interactions compete with other intermolecular forces (dipole-dipole, hydrogen bonding, electrostatic). As a result, these side chains are hydrophobic. Because the “solvent” inside cells is water, this often results in groups of these amino acid side chains clustering together to form “hydrophobic pockets”. This clustering helps generate some of the structural features of proteins/enzymes. The same interaction is also used to help orient molecules inside enzymes for catalysis.

Isoleucine is one of two common amino acids with multiple stereocentres. L-Isoleucine (2S, 3S) is the common amino acid. The other three stereoisomers are rarely found in nature and are not widely used in proteins/enzymes. In general, this additional stereocentre does not play a significant role in biological functions beyond defining the shape(s) that the side chain can adopt.

Proline is the only common amino acid that has a cyclic side chain. This means its amine is secondary rather than primary. The change affects its acidity/basicity and the hydrogen bonding ability of the amine/amide. The ring structure makes the amino acid MUCH less flexible, which leads to it commonly being incorporated in areas that take advantage of this (see Section 4.5.2.4). Finally, the ring structure leads to different amide conformational preferences when incorporated into peptides (see Section 4.4.4.1).

4.2.1.3. Pseudo-Aliphatic (Methionine)

One of the common amino acids has a thioether (sulfide) in its side chain (Figure 4.12). This is technically not an aliphatic group but methionine often behaves similarly to amino acids with aliphatic side chains (see Section 4.2.1.2). It is also semi-commonly found in similar places in proteins/enzymes.

Figure 4.12 – Common Amino Acid with a Thioether Side Chain: L-Methionine.

Carbon and sulfur have identical electronegativities (2.5). Unlike other carbon-heteroatom bonds (e.g. carbon-oxygen and carbon-nitrogen bonds) carbon-sulfur bonds are not polar. The side chain of methionine is not polar and the sulfur functionally behaves as “a large CH₂” in terms of interactions.

4.2.1.4. Special Case (Cysteine)

One of the common amino acids has a thioalcohol (thiol) in its side chain (Figure 4.13).

Figure 4.13 – Common Amino Acid with a Thioalcohol Side Chain: L-Cysteine.

Again, carbon-sulfur bonds are not polar. Sulfur-hydrogen bonds are also not polar. The thioalcohol side chain of methionine is not polar and cannot participate in hydrogen bonding. However, the hydrogen of the thioalcohol is acidic. Because the pK_a of the S-H bond (8.14) is close to standard physiological pH (~7.4) the thioalcohol will often be in equilibrium with its deprotonated form (an anionic sulfur, also called a thiolate; Scheme 4.2). The anionic form is polar, can participate in hydrogen bonding, and may be involved in electrostatic interactions.

Scheme 4.2 – Acid-Base Equilibrium for the Side Chain of L-Cysteine Highlighting the Different Properties of the Two Forms.

Whether the neutral protonated form or the anionic deprotonated form will be more favoured will often depend on the specific local environment (see Section 6.4). As a result, the properties of cysteine can vary significantly: it may behave similarly to amino acids with aliphatic side chains (see Section 4.2.1.2), similarly to amino acids with polar anionic side chains (see Section 4.2.1.8), or alternate between the two.

Cysteine differs from the other common amino acids in that it can form a covalent bond between the sulfur of its side chain and the sulfur of another cysteine’s side chain (Scheme 4.3). This forms a disulfide (R-S-S-R) functional group. These are structurally similar to peroxides but are much more stable.

Scheme 4.3 – Generalized Equilibrium for Disulfide Formation Between the Side Chains of Two L-Cysteines in Peptides.

The mechanism for the formation of the disulfide involves the use of several oxidative enzymes and lies outside the scope of this text. However, the presence of these disulfides is incredibly important for the formation of the complex shapes and structural rigidity of many proteins/enzymes (see Section 4.5.3).

4.2.1.5. Aromatic (Phenylalanine, Tyrosine, Tryptophan, Histidine)

Several of the common amino acids have aromatic groups as side chains (Figure 4.14).

Figure 4.14 – Common Amino Acids with Aromatic Side Chains: L-Phenylalanine, L-Tyrosine, L-Tryptophan, and L-Histidine.

All four of these amino acids are capable of pi-stacking (π-stacking) interactions. These are a special type of noncovalent interaction where the electrons of the pi clouds of aromatic rings attract them together (Figure 4.15). There are several different ways for this to occur: so-called “sandwich” pi-stacking (one ring directly above the other), “T-shaped” pi-stacking (one ring pointing into the other), and/or “parallel displaced” pi-stacking (one ring above but offset from the other).

Figure 4.15 – The Three Common Forms of Pi-Stacking (π-Stacking) Interactions.

The specific details and nature of pi-stacking interactions are complex and still debated by many chemists. However, it is not debated that the presence of pi-stacking interactions is important for the formation of the complex shapes and structural rigidity of many proteins/enzymes (see Section 4.5.3). The same interaction is also used to help orient molecules inside enzymes for catalysis.

In addition to the pi-stacking interactions, each of these side chains has other properties that distinguish them from each other.

Phenylalanine has a side chain that is a non-polar aromatic group. This is technically not an aliphatic group but phenylalanine often behaves similarly to amino acids with aliphatic side chains (see Section 4.2.1.2). It is also semi-commonly found in similar places in proteins/enzymes. The side chain of tryptophan is slightly polar but again tryptophan often behaves similarly to amino acids with aliphatic side chains.

Tyrosine has a side chain that is a polar (hetero)aromatic group. The hydrogen of the alcohol is slightly acidic. Because the pK_a of the O-H bond (10.10) is moderately close to standard physiological pH (~7.4) the alcohol will occasionally be in equilibrium with its deprotonated form. More commonly, it remains protonated and participates in hydrogen bonding (as both a donor and acceptor). In some enzymes the local environment (see Section 6.4) allows it to be involved in catalytic cycles as an acid catalyst.

Histidine is notably different. It is often instead grouped with amino acids that have polar cationic side chains (see Section 4.2.1.7). The side chain is a polar (hetero)aromatic group. The hydrogen of the N-H bond in the side chain can participate in hydrogen bonding as a donor, while the nitrogen without a hydrogen can participate in hydrogen bonding as an acceptor. However, the ring has additional properties that make histidine different from previous amino acid examples.

One difference is that the side chain exists as a pair of tautomers (Scheme 4.4). Recall that tautomers are a pair of constitutional isomers that can readily interconvert. In this case, which nitrogen has the hydrogen changes between the two forms. This can cause confusion in some instances; both forms are still considered histidine. There are multiple ways that the tautomers can interconvert.

Scheme 4.4 – Generalized Equilibrium for Tautomers of the Side Chains of L-Histidine.

One of the ways involves the other notable property of histidine. The nitrogen without a hydrogen of the side chain is moderately basic. Because the pK_a of the conjugate acid (6.04) is close to standard physiological pH (~7.4) the ring will occasionally be in equilibrium with its protonated form (Scheme 4.5).

Scheme 4.5 – Acid-Base Equilibrium for the Side Chain of L-Histidine.

The protonated form has multiple resonance structures that makes both of the N-H bonds of the ring acidic (Scheme 4.6). This effect is one of the ways that the forms interconvert but it is also the primary use of histidine in enzymes; histidine is often involved in catalytic cycles as a base catalyst (often accompanied by this tautomer change). This is in addition to its uses for traditional hydrogen bonding.

Scheme 4.6 – Acid-Base Equilibrium and Tautomer Interconversion for the Side Chain of L-Histidine.

4.2.1.6. Polar, Neutral (Serine, Threonine, Asparagine, Glutamine)

Several of the common amino acids have polar groups as side chains. One set has polar side chains that are normally neutral under physiological conditions (Figure 4.16).

Figure 4.16 – Common Amino Acids with Polar Side Chains that Remain Neutral: L-Serine, L-Threonine, L-Asparagine, and L-Glutamine.

While it is possible to deprotonate these side chains their pK_a values are much higher than previous examples. In biological systems these O-H and N-H groups are not acidic enough to be deprotonated outside of extremely specific conditions.

These amino acids have side chains that are overall polar. They also participate in hydrogen bonding (as both a donor and acceptor). As a result, these side chains are hydrophilic. Because the “solvent” inside cells is water, this often results in groups of these amino acid side chains facing outwards (into the water) to help solvate the chain. The same interaction is also used to help orient molecules inside enzymes for catalysis.

Threonine is one of two common amino acids with multiple stereocentres. L-Threonine (2S, 3R) is the common amino acid. The other three stereoisomers are rarely found in nature and are not widely used in proteins/enzymes. In general, this additional stereocentre does not play a significant role in biological functions beyond defining the shape(s) that the side chain can adopt.

4.2.1.7. Polar, Cationic (Arginine, Lysine)

Several of the common amino acids have polar groups as side chains. One set has polar side chains that are normally cationic under physiological conditions (Figure 4.17).

Figure 4.17 – Common Amino Acids with Polar Side Chains that are Typically Cationic: L-Arginine and L-Lysine.

Because the pK_a value for the conjugate acid of arginine and lysine’s side chains (12.10 and 10.67 respectively) are much higher than standard physiological pH (~7.4) the side chains will almost always exist as their protonated forms (the conjugate acids; Scheme 4.7). For both, the cationic form is still polar, can participate in hydrogen bonding as a donor, and may be involved in electrostatic interactions.

Scheme 4.7 – Equilibria Between the “Parent” Neutral Forms of L-Arginine and L-Lysine and Their Cationic Forms.

As a result, these side chains are hydrophilic. The electrostatic interactions are variously used to help solvate the chain, for the formation of the complex shapes and structural rigidity of many proteins/enzymes (see Section 4.5.3), and/or to help orient molecules inside enzymes for catalysis. These amino acids are occasionally, but rarely, involved in enzymatic catalytic cycles as acid/base catalysts.

4.2.1.8. Polar, Anionic (Aspartic Acid, Glutamic Acid)

Several of the common amino acids have polar groups as side chains. One set has polar side chains that are normally anionic under physiological conditions (Figure 4.18).

Figure 4.18 – Common Amino Acids with Polar Side Chains that are Typically Anionic: L-Aspartic Acid and L-Glutamic Acid.

Because the pK_a values for the acids of aspartic and glutamic acids’ side chains (3.71 and 4.15 respectively) are much lower than standard physiological pH (~7.4) the side chains will almost always exist as their deprotonated forms (the conjugate bases; Scheme 4.8). For both, the anionic form is still polar, can participate in hydrogen bonding as an acceptor, and may be involved in electrostatic interactions.

Scheme 4.8 – Equilibria Between the “Parent” Neutral Forms of L-Aspartic Acid and L-Glutamic Acid and Their Anionic Forms.

As a result, these side chains are hydrophilic. The electrostatic interactions are variously used to help solvate the chain, for the formation of the complex shapes and structural rigidity of many proteins/enzymes (see Section 4.5.3), and/or to help orient molecules inside enzymes for catalysis. These amino acids are occasionally, but rarely, involved in enzymatic catalytic cycles as acid/base catalysts.

4.2.2. Zwitterions and Isoelectric Point (pI)

In general, amino acids exist as their zwitterionic forms (ignoring the side chains) under physiological conditions. However, the actual protonation state (anionic, neutral, cationic) of each of the functional groups in an amino acid will depend on the specific pH of the solution. The isoelectric point (abbreviated as pI or pH(I)) is the pH at which a compound has on average no net ionic charge. For amino acids this means that the major form(s) at equilibrium at this pH is net neutral; the various acid-base reactions are still occurring but the major compound in solution will be the neutral compound (or average to a neutral charge).

Knowing a molecule’s isoelectric point has a variety of applications. Most commonly it is used to help identify an unknown compound and/or to separate similar compounds (electrophoresis). The specific details of how these techniques work are outside the scope of this text and are instead typically explored in introductory biochemistry courses.

4.2.2.1. How to Determine an Amino Acid’s Isoelectric Point (pI)

It is possible to use pK_a values to determine the isoelectric point of a compound. For amino acids there are two methods depending on the nature of the side chain. In both cases this is fundamentally just finding the average between the pK_a values that protonate and deprotonate the neutral compound.

If the amino acid’s side chain IS NOT acidic/basic:

1. Calculate the average of the pK_a values for the ammonium and the carboxylic acid. This is the pI value.

This method is simple but obscures what is actually being calculated. The pI value is the average of the two pK_a values (viz. pH values) that represent formation of the cationic and anionic forms.

If the amino acid’s side chain IS acidic/basic:

1. Draw the molecule as it would exist at a very low pH, with all functional groups in their (conjugate) acid forms.

2. Remove hydrogens from groups one-by-one in ascending order using pK_a values. This is the order that they would be removed in solution (most acidic first, least acidic last). It may be helpful to keep track of the net charge on the molecules.

Calculate the average of the pK_a values for the shift between the cationic and neutral form and the shift from the neutral to the anionic form. This is the pI value.

Because the calculated pI value will depend on the specific pK_a values used different tables may give slightly different results. Unless otherwise indicated, for all pI calculations use the pK_a values specified in this text (Figure 4.3).

Familiarity with the approach will help when calculating pI values for other, more complex, systems (see Section 4.5.1.1).