Amino Acids and the Peptide Bond

Amino acids are organic compounds with an amino group (–NH₂) and a carboxyl group (–COOH) attached to the same α-carbon, and the peptide bond is the amide link formed between the carboxyl group of one amino acid and the amino group of the next, with the loss of water. In brief: glycine (75.07 g/mol) and alanine (89.09 g/mol) join to form the dipeptide glycylalanine (146.14 g/mol), and 20 standard amino acids can form 20¹⁰ ≈ 10¹³ different peptides of length 10.

Proteins, the working molecules of the cell, are built from just 20 common amino acids, arranged in a specific sequence. The properties of the side chains and the nature of the peptide bond determine how a protein folds and what it does. This article describes the structure of amino acids and their charge in solution, classifies the 20 standard amino acids, explains the peptide bond, and shows calculations of isoelectric points and peptide masses, with tables and worked examples.

What is the structure of an amino acid?

The general formula is H₂N–CHR–COOH. The central α-carbon is bonded to four groups: an amino group, a carboxyl group, a hydrogen atom and a variable side chain R. The side chain makes each amino acid different (R = H in glycine, CH₃ in alanine). Since the α-carbon has four different groups in all of the standard amino acids except glycine, they are chiral, and those in proteins are almost always of the L configuration (S in the R/S system, except for cysteine, which is R). D-Amino acids are found in some bacterial cell walls and antibiotics.

Why do amino acids form zwitterions?

The carboxyl group is acidic (pK_a about 2) and the amino group is basic (the pK_a of the conjugate acid is about 9–10). In neutral solution, the acid group has lost a proton and the amino group has gained one, so the molecule is H₃N⁺–CHR–COO⁻, a zwitterion (dipolar ion) with a net charge of zero. This explains why amino acids are crystalline solids with high melting points (often above 200 °C, with decomposition) that dissolve in water but not in nonpolar solvents. In strong acid (pH below 2) the amino acid is a cation (net charge +1), and in strong base (pH above 10) an anion (net charge −1). The pH at which the net charge is zero is the isoelectric point (pI), where the amino acid does not move in an electric field.

How are the 20 amino acids classified?

• Nonpolar (hydrophobic) side chains: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan. They are usually found inside proteins.
• Polar uncharged side chains: serine, threonine, cysteine, tyrosine, asparagine, glutamine. They can form hydrogen bonds. Cysteine can form disulfide bonds (–S–S–) with another cysteine.
• Acidic side chains (negative at pH 7): aspartic acid, glutamic acid.
• Basic side chains (positive at pH 7): lysine, arginine, histidine.

Nine amino acids are essential for adult humans, which means that they must be obtained from food: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.

Examples of amino acids

What is the peptide bond?

When the carboxyl group of one amino acid reacts with the amino group of another, a molecule of water is lost, and an amide bond is formed (the peptide bond): –CO–NH–. The product is a dipeptide, and further additions give tripeptides, oligopeptides (a few residues) and polypeptides (dozens to thousands). A protein is one or more long polypeptides folded into a definite shape. By convention, a peptide is written from the free amino end (N-terminus) to the free carboxyl end (C-terminus): Gly-Ala is glycylalanine, with glycine at the N-terminus.

The peptide bond has a partial double-bond character due to resonance: the C–N bond length is about 133 pm, between a single bond (147 pm) and a double bond (127 pm). As a result, the six atoms of the peptide group (the α-carbons, C, O, N and H) lie in a plane, and rotation about the C–N bond is restricted (a barrier of about 80 kJ/mol). The groups are nearly always in the trans arrangement, except in proline. Rotation can take place about the bonds on either side of the α-carbon (the angles φ and ψ), and this is what lets chains fold. The peptide bond is kinetically stable: in water at neutral pH the half-life is hundreds of years, although enzymes (proteases) hydrolyze it quickly.

How do you calculate the isoelectric point?

For an amino acid with no ionizable side chain, the pI is the average of the two pK_a values: pI = (pK_a1 + pK_a2) / 2. For amino acids with ionizable side chains, take the average of the two pK_a values that surround the neutral form: for acidic amino acids the two lowest, and for basic ones the two highest.

Worked examples

Example 1: pI of glycine

pK_a1 (COOH) = 2.34 and pK_a2 (NH₃⁺) = 9.60. pI = (2.34 + 9.60) / 2 = 5.97.

Example 2: pI of glutamic acid

pK_a values: α-COOH 2.19, side chain COOH 4.25, α-NH₃⁺ 9.67. The neutral form lies between the first two, so pI = (2.19 + 4.25) / 2 = 3.22. At pH 7, glutamic acid has a net charge of −1.

Example 3: pI of lysine

pK_a values: α-COOH 2.18, α-NH₃⁺ 8.95, side chain NH₃⁺ 10.53. The neutral form lies between the last two, so pI = (8.95 + 10.53) / 2 = 9.74. At pH 7, lysine carries a net charge of +1.

Example 4: the mass of a dipeptide and a tripeptide

• Gly–Ala: 75.07 + 89.09 − 18.02 = 146.14 g/mol.
• Gly–Gly–Gly: 3 × 75.07 − 2 × 18.02 = 189.17 g/mol.
• A protein of 300 amino acids: the average mass of a residue (after loss of water) is about 110 g/mol, so M ≈ 33,000 g/mol (33 kDa).

Example 5: the number of possible peptides

A tripeptide can have 20³ = 8000 sequences. A peptide of 10 residues has 20¹⁰ = 1.0 × 10¹³ possible sequences, and a small protein of 100 residues 20¹⁰⁰ ≈ 10¹³⁰, more than the number of atoms in the observable universe (about 10⁸⁰). Only a tiny fraction of them is found in nature.

Example 6: the number of peptides from three amino acids

With three different amino acids (Gly, Ala, Ser), the number of different tripeptides using each once is 3! = 6, for example Gly-Ala-Ser and Ser-Ala-Gly, which are different, because they have different N- and C-termini.

How were amino acids and peptides discovered?

The first amino acid, asparagine, was isolated from asparagus juice in 1806 by Louis Vauquelin and Pierre Robiquet. Glycine was obtained from gelatin by Henri Braconnot in 1820, and its sweet taste gave it the name. Emil Fischer and Franz Hofmeister proposed independently in 1902 that proteins are chains of amino acids linked by peptide bonds, and Fischer made the first synthetic dipeptide, glycylglycine, in 1901. The last of the 20 standard amino acids to be discovered was threonine, in 1935, by William Rose, who also worked out the essential amino acids. Frederick Sanger determined the complete sequence of insulin (51 amino acids) in 1955, for which he received the Nobel Prize in Chemistry in 1958.

Which mistakes are common?

• Drawing amino acids as neutral molecules in water: at pH 7 they are zwitterions.
• Forgetting that a peptide has direction: Gly-Ala differs from Ala-Gly.
• Subtracting too few waters: a chain of n residues has n − 1 peptide bonds.
• Using the wrong pK_a values for pI: use the two values around the neutral form.
• Calling the peptide bond a simple single bond: it is planar with partial double-bond character.