BIOCHEMISTRY 4115: Amino Acids, pKa, pI, and pH

Course Information:

• Course: BIOCHEMISTRY 4115
• Lecture Days/Times: Monday, Wednesday, Friday 11:15 AM – 12:05 PM; Wednesday 5:30 PM – 6:20 PM (usually led by Teaching Assistants)
• Date: August 27, 2025
• Instructor: Dr. Daniel J. Slade (dslade@vt.edu)
• Topic: Amino Acids, pKa, pI, and pH

The 20 Common Amino Acids of Proteins:

• Classification based on side chains:
  • Nonpolar side chains: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M), Phenylalanine (F), Tryptophan (W), Proline (P).
  • Polar side chains: Serine (S), Threonine (T), Cysteine (C), Tyrosine (Y), Asparagine (N), Glutamine (Q).
  • Electrically charged side chains:
    • Acidic: Aspartate (D), Glutamate (E).
    • Basic: Lysine (K), Arginine (R), Histidine (H).

Acid-Base Properties of Amino Acids:

• Amino acids are classified as weak polyprotic acids.
• A polyprotic acid is an acid capable of donating more than one proton ($H^+$) per molecule to an aqueous solution.

Key Definitions:

• pKa:
  • The negative base-10 logarithm of the acid dissociation constant ($K_a$) of a solution.
  • Formula: $pKa = -log<em>{10}K</em>a$
  • Significance: A lower pKa indicates a stronger acid.
  • Reverse Calculation: $K_a = 10^{-pKa}$
  • Critical Point: When $pH = pKa$, $50\%$ of the functional group exists in its acid form and $50\%$ in its conjugate base form.
• pI (Isoelectric Point):
  • The pH at which the net charge of a molecule (e.g., an amino acid or protein) is zero, or neutral.
  • Under this condition, proteins are generally in their least soluble and stable form, a principle important in protein isolation and purification.
• pH:
  • The negative base-10 logarithm of the concentration of protons ($[H^+]$).
  • Formula: $pH = -log_{10}[H^+]$
  • Significance: A lower pH indicates a more acidic solution (due to a higher concentration of $H^+$).
  • Relationship with $[H^+]$ and $[OH^-]$: As $[H^+]$ increases, $[OH^-]$ decreases.

Anatomy of an Amino Acid (at neutral pH):

• Amino acids are tetrahedral structures.
• Key components:
  • $\alpha$-Carbon ($C_\alpha$)
  • Amino group ($-NH_3^+$)
  • Carboxyl group ($-COO^-$)
  • Side chain ($-R$ group)
• Except for proline and its derivatives, all common proteins share this basic structure.

Ionic Forms of Amino Acids - Acid-Base Behavior of Glycine:

• Amino acids change their charge states depending on the pH.
• Example with Glycine (R = H):
  • Cationic form (pH 1, Net charge +1): Both the amino and carboxyl groups are protonated ($H<em>3N^+-CH</em>2-COOH$).
  • Zwitterion (pH 7, Net charge 0): The amino group is protonated ($NH<em>3^+$) and the carboxyl group is deprotonated ($COO^-$) ($H</em>3N^+-CH_2-COO^-$).
  • Anionic form (pH 13, Net charge -1): Both the amino and carboxyl groups are deprotonated ($H<em>2N-CH</em>2-COO^-$).

Titration Curves of Amino Acids:

• All amino acids are polyprotic, meaning they have multiple pKs corresponding to their ionizable groups.
• Glycine (Neutral AA): Has two pKs.
  • $pK_1$ (for the $\alpha$-carboxyl group) is around $2.34$.
  • $pK_2$ (for the $\alpha$-amino group) is around $9.60$.
  • Its isoelectric point (pI) is the average of $pK<em>1$ and $pK</em>2$. For glycine, $pI = \frac{2.34 + 9.60}{2} \approx 5.97$.
• Lysine (Basic AA): Has three pKs.
  • $pK_1$ ($\alpha$-carboxyl)
  • $pK_2$ ($\alpha$-amino)
  • $pK_3$ (R-group amino)
  • Its pI is calculated as the average of the two pKa values that bracket the neutral zwitterionic form (i.e., the $pK_a$ values of the two basic groups for basic amino acids).
• Glutamate (Acidic AA): Has three pKs.
  • $pK_1$ ($\alpha$-carboxyl)
  • $pK_2$ (R-group carboxyl)
  • $pK_3$ ($\alpha$-amino)
  • Its pI is calculated as the average of the two pKa values that bracket the neutral zwitterionic form (i.e., the $pK_a$ values of the two acidic groups for acidic amino acids).

Influence of pH on Amino Acid Charge States:

• Below pH 2: The molecule typically has a +1 charge (cationic form).
• At pH 3-8 (around neutral pH): The molecule is typically neutral (zwitterionic form).
• Above pH 9: The molecule typically has a -1 charge (anionic form).
• Tip: Understanding how to calculate the overall charge at different pHs is crucial.

pKa and Protonation/Deprotonation Rules:

• pH < pKa: The ionizable functional group will be protonated.
• pH > pKa: The ionizable functional group will be deprotonated.
• General pKa values:
  • $\alpha$-carboxyl group: pKa $\approx 2$
  • $\alpha$-amine group: pKa $\approx 9$

The Power of Selenocysteine (example of pKa significance):

• Cysteine pKa: $8.3$
• Selenocysteine pKa: $5.2$
• Question: Which amino acid is the better nucleophile at biological pH? (Answer: Selenocysteine, due to its lower pKa, meaning its -SeH group is more readily deprotonated to -Se- at physiological pH, making it a stronger nucleophile).

Amino Acid Connections: Peptide Bonds and Polypeptides:

• Amino acids are linked by covalent peptide bonds.
• Peptide bonds are formed in a condensation reaction (also called a dehydration reaction) by ribosomes.
• A polypeptide is a long linear chain made of many amino acids (typically $\sim50$ or more AAs).
  • Peptides are shorter ($\sim50$ or fewer AAs).
• Amino acids within a polypeptide molecule are called amino acid residues.
• A polypeptide chain has directionality:
  • N-terminus: The beginning of the chain with a free $\alpha$-amino group (hydrogen bond donor).
  • C-terminus: The end of the chain with a free $\alpha$-carboxyl group (hydrogen bond acceptor).

Calculating the Isoelectric Point (pI) of a Protein:

• The pI of a free amino acid can be different from the pI of a polypeptide or protein.
• The folding of a protein can drastically alter its true pI by burying or exposing ionizable side chains.
• ProtParam Tool: A bioinformatics tool (e.g., on ExPASy) that calculates theoretical pI and other physicochemical parameters for a given protein sequence.
  • Input: Swiss-Prot/TrEMBL accession number or amino acid sequence.
  • Output: Molecular weight, theoretical pI, amino acid composition, etc.
  • Example result: A protein with 479 amino acids and a molecular weight of $50855.38$ Da has a theoretical pI of $8.86$.

Protein Overall Charge:

• The majority ($\sim95\%$) of naturally-occurring proteins have a low net charge.
• Some proteins are highly charged, such as histones.
  • Histones are positively-charged due to a high content (about $24\%$) of basic amino acids like lysine and arginine in their primary structure.
  • This positive charge enables their crucial interactions with the negatively-charged DNA (forming nucleosomes).
  • Chromatin remodeling can be prompted by modifications like acetylation and phosphorylation, which neutralize histone's inherent positive charge and reduce its attraction to the nucleic acid backbone.

$K_w$, the Ion Product of Water:

• Autoionization of Water: Water dissociates into hydronium ions ($H^+$) and hydroxide ions ($OH^-$).
  • Reaction: $H_2O \leftrightarrow H^+ + OH^-$
• Equilibrium Constant ($K_{eq}$) for Water Dissociation:
  • Formula: $K<em>{eq} = \frac{[H^+][OH^-]}{[H</em>2O]}$
• Molarity of Pure Water:
  • Density of water: $1 \ g/mL$ or $1000 \ g/L$
  • Molecular weight of $H_2O$: $18 \ g/mol$
  • Molarity: $\frac{1000 \ g/L}{18 \ g/mol} = 55.5 \ M$
  • Thus, pure water is $55.5 \ M$.
• Derivation of $K_w$:
  • For pure water at $25^{\circ}C$, $[H^+] = [OH^-] = 10^{-7} \ M$
  • $K_{eq} = \frac{(10^{-7})(10^{-7})}{55.5} \approx 1.8 \times 10^{-16} \ M$
  • Since the concentration of $H<em>2O$ is essentially constant in dilute aqueous solutions, a new constant, $K</em>w$ (the ion product of water), is defined:
  • $K_w = [H^+][OH^-]$
  • Given that $K<em>w = 55.5 \times K</em>{eq}$, then $K_w = 55.5 \times (1.8 \times 10^{-16}) \approx 1.0 \times 10^{-14} \ M^2$
  • Therefore: $K_w = [H^+][OH^-] = 10^{-14} \ M^2$ at $25^{\circ}C$.