Amino Acid Structure & Properties

General Formula

The generic structure of an $\alpha$-amino acid can be written as:

At neutral pH, amino acids exist predominantly as zwitterions with the amino group protonated ($-\text{NH}_3^+$) and the carboxyl group deprotonated ($-\text{COO}^-$).

Nonpolar / Hydrophobic (9 amino acids)

These residues have aliphatic or aromatic R-groups that are largely insoluble in water. They tend to cluster in the interior of globular proteins, away from the aqueous environment, stabilizing the tertiary structure through hydrophobic interactions.

Polar Uncharged (6 amino acids)

These amino acids have R-groups capable of hydrogen bonding but carry no formal charge at pH 7.4. They are often found on protein surfaces, mediating interactions with water and other polar molecules.

Positively Charged at pH 7.4 (3 amino acids)

These amino acids carry a net positive charge under physiological conditions.

Negatively Charged at pH 7.4 (2 amino acids)

These amino acids carry a net negative charge under physiological conditions due to deprotonated carboxyl side chains.

Ionization Equilibria

A simple amino acid (no ionizable R-group) undergoes two ionization steps:

The first ionization (p$K_{a1}$) corresponds to loss of a proton from the carboxyl group; the second (p$K_{a2}$) to loss of a proton from the ammonium group.

The Henderson-Hasselbalch Equation

The relationship between pH, p$K_a$, and the ratio of conjugate base to acid is given by:

Key implications:

• When $\text{pH} = \text{p}K_a$, then $[\text{A}^-] = [\text{HA}]$ and the group is 50% ionized
• When $\text{pH} = \text{p}K_a + 1$, the ratio $[\text{A}^-]/[\text{HA}] = 10$ (90.9% deprotonated)
• When $\text{pH} = \text{p}K_a - 1$, the ratio $[\text{A}^-]/[\text{HA}] = 0.1$ (9.1% deprotonated)
• Effective buffering capacity exists within $\pm 1$ pH unit of the p$K_a$

Definition

The isoelectric point (pI) is the pH at which the amino acid carries no net electrical charge. At the pI, the amino acid will not migrate in an electric field (electrophoresis) and has minimum solubility.

Titration of Alanine (2 ionizable groups)

Starting from fully protonated form ($\text{H}_3\text{N}^+ - \text{CHR} - \text{COOH}$, net charge = +1) and adding NaOH:

• 0 to 1 equivalent NaOH: Titration of $\alpha$-COOH (p$K_{a1} = 2.34$). Buffering region near pH 2.34.
• At 0.5 equivalents: pH = p$K_{a1} = 2.34$. Equal amounts of cation and zwitterion.
• At 1 equivalent: pH = pI = $\frac{2.34 + 9.69}{2} = 6.02$. Pure zwitterion form.
• 1 to 2 equivalents NaOH: Titration of $\alpha$-$\text{NH}_3^+$ (p$K_{a2} = 9.69$). Buffering region near pH 9.69.
• At 1.5 equivalents: pH = p$K_{a2} = 9.69$. Equal amounts of zwitterion and anion.
• At 2 equivalents: Fully deprotonated anion ($\text{H}_2\text{N} - \text{CHR} - \text{COO}^-$, net charge = -1).

Titration of Glutamic Acid (3 ionizable groups)

Glutamic acid has three p$K_a$ values corresponding to the $\alpha$-COOH (p$K_{a1} = 2.19$), $\gamma$-COOH (p$K_{aR} = 4.25$), and $\alpha$-$\text{NH}_3^+$ (p$K_{a2} = 9.67$).

The titration curve shows three buffering plateaus and requires 3 equivalents of NaOH for complete titration.

Note: for acidic amino acids, the pI falls between the two lowest p$K_a$ values (both carboxyl groups). For basic amino acids, the pI falls between the two highest p$K_a$ values (both amino groups or amino + imidazole).

Buffering Capacity

The buffering capacity $\beta$ is defined as the amount of strong acid or base required to change the pH by one unit. Mathematically:

where $C_T$ is the total buffer concentration. Buffering capacity is maximal when $\text{pH} = \text{p}K_a$ (i.e., when $[\text{H}^+] = K_a$).

Proline -- The Cyclic Imino Acid

Proline is unique among the 20 standard amino acids because its side chain cyclizes back onto the backbone nitrogen, forming a five-membered pyrrolidine ring. This makes proline technically an imino acid rather than an amino acid, as the nitrogen bears only one hydrogen in its free form.

• The cyclic structure restricts the backbone dihedral angle $\phi$ to approximately $-65°$
• Proline introduces kinks in $\alpha$-helices and is often found in turns
• The $\text{X-Pro}$ peptide bond has a higher propensity for the cis configuration (~10%) compared to other peptide bonds (<0.1%)
• Proline residues are important in collagen, where every third residue is Gly and Pro/Hyp are abundant

Cysteine -- Disulfide Bond Formation

Cysteine's thiol group ($-\text{SH}$, p$K_a \approx 8.3$) can form covalent disulfide bonds ($-\text{S}-\text{S}-$) with another cysteine via oxidation:

The resulting cystine residue is critical for stabilizing the tertiary and quaternary structure of many extracellular proteins (e.g., insulin, immunoglobulins). The standard reduction potential is:

Glycine -- The Simplest Amino Acid

Glycine ($\text{R} = \text{H}$) is the smallest amino acid and the only one that is achiral. Its small size gives it maximal conformational flexibility in proteins:

• Glycine occupies the widest region of the Ramachandran plot
• It is essential in tight turns and in proteins requiring compact packing (e.g., collagen: Gly-X-Y repeat)
• Glycine also serves as a neurotransmitter and a precursor to porphyrins, purines, and glutathione

Histidine -- The Physiological Buffer

Histidine's imidazole side chain has a p$K_a \approx 6.0$, making it the only standard amino acid whose R-group can ionize near physiological pH. This property makes histidine uniquely suited for several roles:

• Physiological buffering: effective buffer in the pH range 5.0-7.0
• Enzyme catalysis: the imidazole ring can act as both proton donor and acceptor (general acid-base catalysis)
• Metal coordination: the nitrogen atoms of the imidazole ring coordinate metal ions (e.g., $\text{Zn}^{2+}$ in carbonic anhydrase, $\text{Fe}^{2+}$ in hemoglobin)

The protonation equilibrium of the imidazole side chain:

At pH 7.4, approximately $\frac{1}{1 + 10^{7.4 - 6.0}} = \frac{1}{1 + 25.1} \approx 3.8\%$ of histidine residues are protonated. This partial protonation at physiological pH is precisely what makes histidine so versatile in enzyme active sites.