Proteins and Enzymes

• The general structure of amino acids (H₂N—CHR—COOH) and the zwitterion form at physiological pH
• How to classify amino acids by their R group: acidic, basic, neutral, aromatic
• Peptide bond formation by condensation and properties of the —CO—NH— linkage
• The four levels of protein structure: primary, secondary, tertiary, quaternary
• The difference between fibrous and globular proteins with examples
• Denaturation — what it is, what causes it, and why it matters
• Enzyme catalysis: lock-and-key model, active site, inhibition types, optimum conditions

Amino Acids — Building Blocks of Proteins

An amino acid has both an amino group (—NH₂) and a carboxyl group (—COOH) attached to the same carbon atom (the α-carbon). The general structure is:

        H
        |
  H₂N — C — COOH
        |
        R

The R group (side chain) determines the identity and properties of the amino acid.

Zwitterion (dipolar ion) form at physiological pH (~7.4):

        H
        |
  ⁺H₃N — C — COO⁻
        |
        R

At physiological pH, the —NH₂ group is protonated (—NH₃⁺) and the —COOH group is deprotonated (—COO⁻). The molecule is electrically neutral overall but carries both charges — called a zwitterion.

Isoelectric point (pI): The pH at which the amino acid has zero net charge (exists fully as zwitterion). At pH < pI → cation form; at pH > pI → anion form.

Classification of Amino Acids

Essential amino acids (cannot be synthesised by the body; must come from diet): Mnemonic: PVT TIM HaLL — Phenylalanine, Valine, Threonine, Tryptophan, Isoleucine, Methionine, Histidine, Arginine, Leucine, Lysine

Glycine (R = H) is the simplest amino acid — it is the only amino acid with no chiral centre.

Peptide Bond Formation

When the —COOH of one amino acid reacts with the —NH₂ of another, a peptide bond (—CO—NH—) forms with loss of one water molecule (condensation reaction):

H₂N—CHR₁—COOH  +  H₂N—CHR₂—COOH
                          ↓ (−H₂O)
H₂N—CHR₁—CO—NH—CHR₂—COOH
              ↑
         peptide bond

Properties of the peptide bond:

• Partial double bond character (resonance with C=O group) → planar, rigid
• Trans configuration preferred (bulky R groups on opposite sides)
• Cannot freely rotate — restricts protein conformation

Nomenclature:

• 2 amino acids → dipeptide
• 3 → tripeptide
• Many → polypeptide or protein (roughly >50 amino acids)

Levels of Protein Structure

Primary Structure

• The linear sequence of amino acids in the polypeptide chain
• Held together by peptide bonds (covalent)
• Determines all higher levels of structure
• Example: Insulin — A chain (21 aa) + B chain (30 aa) linked by disulfide bridges

Secondary Structure The local spatial arrangement of the polypeptide backbone:

(a) α-Helix:

• Right-handed coil; each turn = 3.6 amino acids; pitch = 5.4 Å
• Stabilised by intramolecular H-bonds between C=O of residue n and N—H of residue n+4
• R groups project outward
• Example: Hair (keratin), myoglobin

(b) β-Pleated Sheet:

• Extended polypeptide chains running parallel or antiparallel
• Stabilised by intermolecular H-bonds between C=O and N—H of adjacent strands
• Example: Silk fibroin

Tertiary Structure

• The overall 3D folding of the entire polypeptide chain
• Stabilised by:
  1. Disulfide bonds (—S—S—) between cysteine residues (strongest; covalent)
  2. Hydrophobic interactions (nonpolar R groups cluster away from water)
  3. Ionic interactions (between acidic and basic R groups; "salt bridges")
  4. H-bonds (between polar R groups)
• Determines the protein's functional shape

Quaternary Structure

• Association of two or more polypeptide chains (subunits) into a functional protein
• Subunits held by same non-covalent interactions as tertiary structure
• Example: Haemoglobin — 4 subunits (2α + 2β), each carrying an O₂-binding haem group

Fibrous vs Globular Proteins

Denaturation

Denaturation is the loss of secondary, tertiary, and/or quaternary structure of a protein without breaking the primary structure (peptide bonds remain intact).

Causes of denaturation:

• Heat (disrupts H-bonds and hydrophobic interactions)
• Extreme pH (disrupts ionic interactions and H-bonds)
• Urea/guanidinium chloride (disrupts H-bonds by competing for donors/acceptors)
• Organic solvents (disrupt hydrophobic interactions)
• Heavy metal salts (form bonds with —SH, —COO⁻ groups)

Result: Loss of biological activity. Example: cooking an egg (albumin denatures → white coagulates); alcohol-based hand sanitiser (denatures bacterial proteins).

Renaturation (refolding): Some proteins can refold spontaneously if the denaturing agent is removed — proving that the primary sequence carries all the information needed for folding (Anfinsen's dogma).

Ramachandran Plot — Peptide Backbone Angles

The conformation of a polypeptide is defined by two backbone torsion angles: φ (phi) around N—Cα bond and ψ (psi) around Cα—C bond. The Ramachandran plot shows which φ,ψ combinations are sterically allowed. α-Helices and β-sheets cluster in distinct allowed regions — this is why not all sequences can form the same secondary structure.

Disulfide Bond Chemistry

Cysteine residues can oxidise to form a disulfide bridge (—S—S—):

2 R—SH → R—S—S—R + 2H⁺ + 2e⁻   (oxidation)

Disulfide bonds are the only covalent cross-links in tertiary/quaternary structure (apart from peptide bonds). They are reduced by reagents like β-mercaptoethanol or DTT. This is exploited in hair-perming (break —S—S— with reducer, reshape, re-oxidise).

Enzyme Catalysis — Detailed Mechanism

Enzymes are biological catalysts (mostly proteins, some RNA = ribozymes). They:

• Lower the activation energy (Eₐ) without being consumed
• Are highly specific (one enzyme catalyses one reaction type)
• Are reusable

Lock-and-key model (Fischer, 1894):

• The enzyme's active site has a rigid shape complementary to the substrate
• Substrate (key) fits exactly into the active site (lock)
• Enzyme–substrate complex (ES) forms → products released → enzyme regenerated

Induced-fit model (Koshland, 1958): The active site is not perfectly rigid; it changes shape slightly to optimally bind the substrate. More accurate model.

Michaelis-Menten equation (JEE awareness level):

Rate (v) = Vmax[S] / (Km + [S])

• Km (Michaelis constant) = [S] at half-maximum velocity → measures enzyme-substrate affinity
• Low Km = high affinity; High Km = low affinity

Enzyme Inhibition:

Example — competitive inhibition: Malonate inhibits succinate dehydrogenase (resembles succinate). Used as pesticide model.

Optimum conditions:

• pH: Each enzyme has an optimal pH (e.g., pepsin pH ~2, salivary amylase pH ~7, trypsin pH ~8)
• Temperature: Rate increases up to optimum (~37°C for human enzymes); above it, denaturation occurs → rate drops sharply

Cofactors and Coenzymes

• Cofactor: Non-protein component required for enzyme activity
  • Inorganic: Metal ions (Fe²⁺ in catalase, Zn²⁺ in carbonic anhydrase)
  • Organic (coenzyme): Small organic molecule (e.g., NAD⁺, FAD, coenzyme A, vitamins)
• Apoenzyme: Protein part of the enzyme (inactive alone)
• Holoenzyme: Apoenzyme + cofactor (fully active)

Example 1: Drawing a Dipeptide and Identifying the Peptide Bond

Problem: Write the structure of the dipeptide Gly-Ala (glycine followed by alanine).
Identify the peptide bond, N-terminus, and C-terminus.

Glycine: H₂N—CH₂—COOH   (R = H)
Alanine: H₂N—CH(CH₃)—COOH  (R = CH₃)

Condensation: —COOH of Gly + H₂N— of Ala → —CO—NH— + H₂O

Gly-Ala dipeptide:

H₂N—CH₂—CO—NH—CH(CH₃)—COOH
  ↑             ↑              ↑
N-terminus  peptide bond   C-terminus

Notes:
• The N-terminus (free —NH₂) is always written on the LEFT by convention.
• The C-terminus (free —COOH) is on the RIGHT.
• Gly-Ala and Ala-Gly are DIFFERENT dipeptides (different primary structure).
• The partial double bond character of —CO—NH— makes it PLANAR.

Example 2: Enzyme Inhibition — Competitive vs Non-Competitive

Problem: An enzyme has Vmax = 100 μmol/min and Km = 5 mM.
Inhibitor X increases apparent Km to 15 mM without changing Vmax.
Inhibitor Y decreases Vmax to 50 μmol/min without changing Km.
Classify each inhibitor and suggest what happens in each case.

Inhibitor X:
  Vmax unchanged, Km increased (apparent affinity decreased)
  → COMPETITIVE inhibitor
  → Binds active site; competes with substrate
  → Adding more substrate can outcompete the inhibitor and restore full activity

Inhibitor Y:
  Km unchanged, Vmax decreased
  → NON-COMPETITIVE inhibitor
  → Binds allosteric site (away from active site)
  → Causes conformational change; substrate can still bind but ES complex is less productive
  → Adding more substrate does NOT restore full Vmax

Key rule: If Km changes → competitive; if Vmax changes → non-competitive.

1. Draw the zwitterion form of alanine (R = —CH₃) at pH 7.
2. How many peptide bonds are present in a polypeptide of 50 amino acids?
3. Name the four types of interactions that stabilise tertiary protein structure.
4. Haemoglobin has quaternary structure. What does this mean, and what is the subunit composition?
5. (Stretch) The enzyme urease is inhibited by Ag⁺ ions. Explain the type of inhibition and the molecular basis. Would adding more substrate (urea) restore enzyme activity? Justify your answer.

• NCERT Class 12 Chemistry, Chapter 14 — Biomolecules, Sections 14.4–14.5
• JEE Foundation: 1–2 questions on protein levels, peptide bonds, enzyme inhibition types
• Common MCQ formats: identify primary/secondary/tertiary/quaternary structure, classify inhibition from Km/Vmax data

Study strategy: Memorise the 4 structural levels with their stabilising forces and one example each. For enzymes, practise classifying inhibition from data tables — competitive vs non-competitive is a high-yield MCQ topic.

Practice MCQs on protein structure levels and enzyme inhibition in the Biomolecules — Proteins and Enzymes topic bank. Try Medium difficulty for JEE Foundation level.

Struggling to remember which level of protein structure involves disulfide bonds vs H-bonds? Ask the Drishti AI tutor to explain with a table of forces vs structure levels.

Complete the Quick Check questions and mark them in your Drishti progress tracker. Aim for 4/5 before moving to nucleic acids.

• Read: Biomolecules — Nucleic Acids — DNA double helix, base pairing, RNA types, central dogma
• Then: Vitamins and Hormones overview
• Practice: Mixed Biomolecules MCQs (Medium difficulty)