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Biochemistry Unit 2: Amino Acids and Peptides

Amino acids are the building blocks of proteins, and their side chains decide how proteins fold, bind and catalyze. This unit covers their structures, charges at different pH values, and how they link into peptides.

Covers: Amino acid structure and stereochemistry, Side-chain classification, Acid-base properties and titration curves, Isoelectric point, The peptide bond, Peptide charge and sequence, Disulfides and side-chain modifications

Study guide

Each standard amino acid has a central α-carbon bonded to an amino group, a carboxyl group, a hydrogen and a side chain (R group). Except for glycine, whose R group is H, the α-carbon is chiral. Proteins use L-amino acids; in CIP terms most are S, but L-cysteine is R because its sulfur changes the priorities. Threonine and isoleucine have a second stereocenter in the side chain. At physiological pH, free amino acids exist as zwitterions, with a protonated –NH₃⁺ and a deprotonated –COO⁻.

Side chains fall into groups. Nonpolar aliphatic: Gly, Ala, Val, Leu, Ile, Met and Pro. Aromatic: Phe, Tyr and Trp, which absorb UV light near 280 nm (mostly Trp and Tyr). Polar uncharged: Ser, Thr, Cys, Asn and Gln. Acidic (negative at pH 7): Asp and Glu. Basic (positive at pH 7): Lys, Arg and His, though His is only partly protonated because its pKa is near 6. Proline is special because its side chain loops back onto its own amino nitrogen, making it rigid and a breaker of α-helices.

Each ionizable group has a pKa. Typical values: α-COOH about 2, α-NH₃⁺ about 9 to 10, Asp and Glu side chains about 4, His about 6, Cys about 8.3, Tyr about 10, Lys about 10.5 and Arg about 12.5. A titration curve shows one flat buffering region per group, centered at its pKa. The isoelectric point (pI) is the pH at which the average net charge is zero. For a simple amino acid, average the two pKa values. For acidic amino acids, average the two lowest; for basic ones, average the two highest. Below its pI a molecule is net positive; above it, net negative.

A peptide bond is an amide that links the carboxyl of one amino acid to the amino group of the next, releasing water. Resonance gives the C–N bond partial double-bond character, so the peptide group is planar and nearly always trans. Rotation is possible only around the bonds to the α-carbon (φ and ψ). Peptides are written from the free amino N-terminus to the free carboxyl C-terminus. To find the net charge of a peptide at a given pH, count both termini plus every ionizable side chain, using partial charges when the pH is near a pKa.

Side chains can be modified after translation. Two cysteines can be oxidized to form a covalent disulfide bond, which stabilizes extracellular proteins; reducing agents such as β-mercaptoethanol or DTT break them. Kinases add phosphate to Ser, Thr or Tyr hydroxyls, adding negative charge that can switch protein activity on or off. Sequence can be determined by Edman degradation, which removes and identifies residues one at a time from the N-terminus, and by mass spectrometry. These tools let biochemists link sequence to structure and function.

Key ideas

• All standard amino acids except glycine are chiral, and proteins use L-amino acids.
• Side chains are grouped as nonpolar, aromatic, polar uncharged, acidic or basic.
• Each ionizable group is protonated below its pKa and deprotonated above it.
• The pI is the average of the two pKa values on either side of the neutral form.
• Peptide bonds are planar and rigid because of amide resonance.
• Net peptide charge equals the sum of terminal and side-chain charges at the given pH.
• Disulfide bonds form by oxidation of two Cys and are broken by reducing agents.

Formulas and rules

Common mistakes

• Averaging all three pKa values to get the pI of an amino acid with an ionizable side chain.
• Forgetting the N- and C-terminal charges when counting a peptide's net charge.
• Assuming histidine is fully positive at pH 7.4, when only a few percent is protonated.
• Calling a disulfide bond formation a reduction rather than an oxidation.
• Writing or reading peptide sequences from the C-terminus instead of the N-terminus.

Before the test, make sure you can

• Identify each standard amino acid's side-chain class from its name or structure.
• Explain why glycine is achiral and why proteins use L-amino acids.
• Predict whether a group is protonated at a given pH from its pKa.
• Calculate the pI of neutral, acidic and basic amino acids.
• Calculate the net charge of a peptide at a given pH.
• Explain why the peptide bond is planar and how peptides are named.
• Calculate peptide masses, accounting for water lost.
• Explain how disulfides form and break, and interpret reducing versus nonreducing gels.

Words to know

α-carbon

The central carbon of an amino acid, bonded to the amino group, carboxyl group, H and side chain.

Zwitterion

A molecule with both positive and negative charges but net charge zero, as amino acids are near neutral pH.

L-amino acid

The stereoisomer used in proteins; most are S, but L-cysteine is R.

Nonpolar side chains

Gly, Ala, Val, Leu, Ile, Met and Pro; usually buried inside proteins.

Aromatic side chains

Phe, Tyr and Trp; Tyr and Trp give proteins their 280 nm absorbance.

Acidic amino acids

Asp and Glu; their carboxylate side chains are negative at pH 7.

Basic amino acids

Lys, Arg and His; Lys and Arg are positive at pH 7, His is partly protonated.

Isoelectric point (pI)

The pH at which a molecule's average net charge is zero.

Peptide bond

An amide bond between the α-carboxyl of one residue and the α-amino of the next.

Amide resonance

Delocalization that gives the peptide C–N bond partial double-bond character, making it planar.

N-terminus

The end of a peptide with a free α-amino group; written first.

C-terminus

The end of a peptide with a free α-carboxyl group; written last.

Disulfide bond

A covalent S–S link formed by oxidizing two cysteine thiols.

Cystine

The name for two cysteines joined by a disulfide bond.

Phosphorylation

Addition of a phosphate to Ser, Thr or Tyr by a kinase, often regulating activity.

Edman degradation

A method that removes and identifies one amino acid at a time from the N-terminus.

Proline

A cyclic amino acid whose ring includes the backbone N; it disrupts α-helices.

Sample practice questions

30 more questions like these, from level 1 to 5, are in the practice set.