Amino Acid Structure
Properties & $\text{p}K_a$ Behavior

Explore the building blocks of proteins: how amino acids are structured, how their ionizable groups respond to pH, and why $\text{p}K_a$ values matter in biochemistry.

Biochemistry

What Is an Amino Acid?

Amino acids are organic molecules that serve as the monomers of proteins. Every amino acid shares a common backbone but carries a unique side chain (R group) that determines its chemical personality.

The Zwitterion: A Molecular Dipole

At physiological pH (~7.4), amino acids exist as zwitterions: molecules with both a positive and a negative charge, yet an overall net charge of zero.

Why Does $\text{p}K_a$ Matter?

The $\text{p}K_a$ of a group tells you the pH at which that group is 50% protonated and 50% deprotonated. It determines how an amino acid behaves as the pH changes.

Low $\text{p}K_a$

The group loses its proton easily: it is a stronger acid. Example: carboxyl group $\text{p}K_a \approx 2$.

High $\text{p}K_a$

The group holds onto its proton tightly: it is a weaker acid. Example: amino group $\text{p}K_a \approx 9$.

The Henderson-Hasselbalch Equation

This equation is the quantitative link between pH, $\text{p}K_a$, and the ratio of protonated to deprotonated forms of an ionizable group:

1

When pH = $\text{p}K_a$: the log term is 0 because $[\text{A}^-] = [\text{HA}]$. The group is exactly half-protonated.

2

When pH > $\text{p}K_a$: $[\text{A}^-] > [\text{HA}]$. The deprotonated form dominates.

3

When pH < $\text{p}K_a$: $[\text{HA}] > [\text{A}^-]$. The protonated form dominates.

Titration Curve of Glycine

$\text{p}K_{a1}$ vs $\text{p}K_{a2}$ vs $\text{p}K_{aR}$

	$\text{p}K_{a1}$	$\text{p}K_{a2}$	$\text{p}K_{aR}$
Group	$-\text{COOH}$	$-\text{NH}_3^+$	R group
Typical Range	1.8 − 2.5	8.8 − 10.5	3.7 − 12.5
Loses Proton	First (lowest pH)	Second	Varies by residue
Present in	All amino acids	All amino acids	Only 7 of 20

The Ionizable Side Chains

Acidic

Asp ($\text{p}K_{aR} \approx 3.7$)
Glu ($\text{p}K_{aR} \approx 4.1$)
Negative at pH 7

Basic

Lys ($\text{p}K_{aR} \approx 10.5$)
Arg ($\text{p}K_{aR} \approx 12.5$)
Positive at pH 7

Special

His ($\text{p}K_{aR} \approx 6.0$)
Cys ($\text{p}K_{aR} \approx 8.3$)
Tyr ($\text{p}K_{aR} \approx 10.1$)

Calculating the Isoelectric Point (pI)

The isoelectric point (pI) is the pH at which the amino acid carries zero net charge. At the pI, the molecule will not migrate in an electric field.

1

Nonpolar / polar (no ionizable R): Average the two backbone $\text{p}K_a$ values: $$\text{pI} = \frac{\text{p}K_{a1} + \text{p}K_{a2}}{2}$$

2

Acidic R group (Asp, Glu): Average the two lowest $\text{p}K_a$ values: $$\text{pI} = \frac{\text{p}K_{a1} + \text{p}K_{aR}}{2}$$

3

Basic R group (Lys, Arg, His): Average the two highest $\text{p}K_a$ values: $$\text{pI} = \frac{\text{p}K_{a2} + \text{p}K_{aR}}{2}$$

How to Determine Net Charge at Any pH

1

List all ionizable groups and their $\text{p}K_a$ values: $\alpha$-COOH, $\alpha\text{-NH}_3^+$, and R group (if applicable).

2

Compare each $\text{p}K_a$ to the pH. If $\text{pH} > \text{p}K_a$, the group has lost its proton (deprotonated). If $\text{pH} < \text{p}K_a$, the group retains its proton (protonated).

3

Assign charges: deprotonated COOH → $COO^-$ (−1), protonated $\text{NH}_2 \rightarrow \text{NH}_3^+$ (+1). Sum all charges.

4

Add up the charges from every group to find the net charge on the entire molecule.

Example: Glycine at pH 1

Glycine has $\text{p}K_{a1} = 2.34$ (COOH) and $\text{p}K_{a2} = 9.60$ ($\text{NH}_3^+$). What is its charge at pH 1?

Example: Glycine at pH 6

Same glycine: $\text{p}K_{a1} = 2.34$, $\text{p}K_{a2} = 9.60$. Now the pH is between the two $\text{p}K_a$ values.

Example: Aspartate at pH 7

Aspartate has three ionizable groups: $\text{p}K_{a1} = 2.1$ (COOH), $\text{p}K_{aR} = 3.9$ (side chain COOH), $\text{p}K_{a2} = 9.8$ ($\text{NH}_3^+$).

Classifying by Side Chain Properties

Nonpolar

Hydrophobic: Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Gly. Found in protein interiors.

Polar Uncharged

Hydrophilic: Ser, Thr, Asn, Gln. Form H-bonds but carry no charge at pH 7.

Negatively Charged

Asp (−), Glu (−). Acidic side chains are deprotonated at pH 7.

Positively Charged

Lys (+), Arg (+), His (+/0). Basic side chains are protonated at pH 7.

Common Mistakes to Avoid

✕ Confusing pH > pKa Direction

When pH > pKa the group is deprotonated, not protonated. Many students reverse this critical rule.

✕ Wrong pKa Pair for pI

For acidic amino acids, average the two lowest pKa values. For basic, average the two highest. Do not always use pKa$_1$ and pKa$_2$.

✕ Forgetting the R Group

Seven amino acids have ionizable R groups. Ignoring the third pKa leads to wrong net charges and wrong pI calculations.

Summary: Key Amino Acids & Their $\text{p}K_a$ Values

Amino Acid	$\text{p}K_{a1}$	$\text{p}K_{aR}$	$\text{p}K_{a2}$	pI
Glycine	2.34	—	9.60	5.97
Alanine	2.35	—	9.87	6.11
Aspartate	2.10	3.86	9.82	2.98
Glutamate	2.10	4.07	9.47	3.08
Histidine	1.77	6.10	9.18	7.64
Lysine	2.18	10.53	8.95	9.74
Arginine	2.01	12.48	9.04	10.76

Question 1 of 5

At physiological pH (7.4), what is the net charge on glycine ($\text{p}K_{a1} = 2.34$, $\text{p}K_{a2} = 9.60$)?

At pH 7.4, pH > $\text{p}K_{a1}$ so COOH is deprotonated (−1), and pH < $\text{p}K_{a2}$ so $\text{NH}_3^+$ remains protonated (+1). Net charge: −1 + 1 = 0 (zwitterion form).

Question 2 of 5

Which amino acid has a side chain $\text{p}K_a$ near physiological pH, making it an effective proton shuttle in enzyme active sites?

Histidine's $\text{p}K_{aR} \approx 6.0$ is closest to physiological pH (~7.4), meaning its imidazole ring can readily accept and donate protons under biological conditions.

Question 3 of 5

What is the isoelectric point (pI) of aspartate given $\text{p}K_{a1} = 2.1$, $\text{p}K_{aR} = 3.9$, $\text{p}K_{a2} = 9.8$?

Aspartate has an acidic R group, so we average the two lowest $\text{p}K_a$ values: $\text{pI} = (2.1 + 3.9) / 2 = 3.0$. A common mistake is averaging $\text{p}K_{a1}$ and $\text{p}K_{a2}$, which gives the wrong answer.

Question 4 of 5

At pH 11, what is the net charge on lysine ($\text{p}K_{a1} = 2.2$, $\text{p}K_{a2} = 9.0$, $\text{p}K_{aR} = 10.5$)?

At pH 11: pH > $\text{p}K_{a1}$ (2.2) so $COO^-$ = −1. pH > $\text{p}K_{a2}$ (9.0) so $\text{NH}_2$ = 0 (deprotonated). pH > $\text{p}K_{aR}$ (10.5) so R-$\text{NH}_2$ = 0 (deprotonated). Net = −1 + 0 + 0 = −1.

Question 5 of 5

According to the Henderson-Hasselbalch equation, when $\text{pH} = \text{p}K_a$, what is the ratio of $[\text{A}^-]$ to $[\text{HA}]$?

When $\text{pH} = \text{p}K_a$, the log term equals zero: $\log([\text{A}^-]/[\text{HA}]) = 0$, which means $[\text{A}^-]/[\text{HA}] = 10^0 = 1$. The group is exactly half protonated and half deprotonated.

Amino Acid Structure
Properties & \(\text{p}K_a\) Behavior

What Is an Amino Acid?

The Zwitterion: A Molecular Dipole

Why Does \(\text{p}K_a\) Matter?

The Henderson-Hasselbalch Equation

Titration Curve of Glycine

\(\text{p}K_{a1}\) vs \(\text{p}K_{a2}\) vs \(\text{p}K_{aR}\)

The Ionizable Side Chains

Calculating the Isoelectric Point (pI)

How to Determine Net Charge at Any pH

Example: Glycine at pH 1

Example: Glycine at pH 6

Example: Aspartate at pH 7

Classifying by Side Chain Properties

Common Mistakes to Avoid

Summary: Key Amino Acids & Their \(\text{p}K_a\) Values

Question 1 of 5

Question 2 of 5

Question 3 of 5

Question 4 of 5

Question 5 of 5

	\(\text{p}K_{a1}\)	\(\text{p}K_{a2}\)	\(\text{p}K_{aR}\)
Group	\(-\text{COOH}\)	\(-\text{NH}_3^+\)	R group
Typical Range	1.8 − 2.5	8.8 − 10.5	3.7 − 12.5
Loses Proton	First (lowest pH)	Second	Varies by residue
Present in	All amino acids	All amino acids	Only 7 of 20

Amino Acid StructureProperties & \(\text{p}K_a\) Behavior

What Is an Amino Acid?

The Zwitterion: A Molecular Dipole

Why Does \(\text{p}K_a\) Matter?

The Henderson-Hasselbalch Equation

Titration Curve of Glycine

\(\text{p}K_{a1}\) vs \(\text{p}K_{a2}\) vs \(\text{p}K_{aR}\)

The Ionizable Side Chains

Calculating the Isoelectric Point (pI)

How to Determine Net Charge at Any pH

Example: Glycine at pH 1

Example: Glycine at pH 6

Example: Aspartate at pH 7

Classifying by Side Chain Properties

Common Mistakes to Avoid

Summary: Key Amino Acids & Their \(\text{p}K_a\) Values

Question 1 of 5

Question 2 of 5

Question 3 of 5

Question 4 of 5

Question 5 of 5

Amino Acid Structure
Properties & \(\text{p}K_a\) Behavior