Explain specificity of enzymes: complementary shape and fit of the active site with the substrate.

5.1 Enzymes – Specificity of Enzymes

Learning objective (AO1)

Explain why enzymes are specific for particular substrates by describing the complementary shape and fit of the enzyme’s active site.

Core definitions (AO1)

• Enzyme: a biological catalyst that accelerates a chemical reaction, is not consumed in the reaction and can be re‑used.
• Active site: a specialised region on the enzyme where the substrate binds.
• Substrate: the reactant that fits into the active site.
• Enzyme‑substrate complex: the temporary association formed when a substrate is bound; after the reaction the enzyme is released unchanged.
• Denaturation: loss of the enzyme’s three‑dimensional shape, usually caused by extreme temperature or pH, rendering the active site non‑functional.

Key terminology (AO1)

How enzymes work – the shape‑fit principle

1. Lock‑and‑key model

• The active site and substrate have exactly matching shapes, like a key fitting a lock.
• No change in enzyme shape is required for binding.

2. Induced‑fit model

• Initial binding of the substrate induces a slight conformational change in the enzyme.
• The change improves the fit and positions catalytic residues for the reaction.

3. Non‑covalent interactions that stabilise the complex

• Hydrogen bonds
• Ionic (electrostatic) bonds
• Van der Waals forces
• Hydrophobic interactions

Why specificity arises

1. The linear sequence of amino‑acids folds into a unique three‑dimensional structure.
2. This folding creates an active site with a precise arrangement of functional groups (–OH, –NH₂, –COOH, –SH, etc.).
3. The spatial arrangement of these groups is complementary to the shape, charge distribution and functional groups of one (or a few) substrate(s).
4. Only substrates that match the active site can form the enzyme‑substrate complex; others are excluded.

Illustrative comparison of lock‑and‑key vs. induced‑fit

Examples of enzyme specificity

• Amylase (salivary/pancreatic) – recognises α‑1,4‑glycosidic bonds in starch.
  Reaction: Starch + H₂O → Maltose
• Pepsin (stomach) – hydrolyses peptide bonds in proteins, optimally at pH ≈ 2.
  Reaction: Protein + H₂O → Peptides
• Lactase (small intestine) – cleaves the β‑1,4‑glycosidic bond in lactose.
  Deficiency → lactose intolerance (a common human‑health application).

Factors influencing enzyme activity (AO1 & AO2)

Temperature

As temperature rises, molecular collisions increase, so activity rises sharply up to an optimum. Beyond this optimum the enzyme denatures and activity falls dramatically.

pH

Each enzyme has an optimum pH that reflects the ionisation state of active‑site residues. Deviations reduce activity; extreme pH values cause denaturation.

Inhibitors

• Competitive inhibitor: resembles the substrate and occupies the active site, reducing the number of available sites.
• Non‑competitive inhibitor: binds elsewhere, altering the enzyme’s shape and lowering activity.

Genetic mutations

Changes in the amino‑acid sequence can alter the shape or charge of the active site, thereby changing substrate specificity or abolishing activity.

Typical syllabus examples

• Amylase – optimum ≈ 37 °C, optimum pH ≈ 7.0.
• Pepsin – optimum ≈ 37 °C, optimum pH ≈ 2.0.
• Lactase – optimum ≈ 37 °C, optimum pH ≈ 6.0‑7.0.

Practical investigation (AO2) – effect of temperature on amylase activity

1. Label five test tubes A–E. Add 5 mL of 1 % starch solution and 0.5 mL of amylase to each tube.
2. Place the tubes in water baths set at 10 °C, 20 °C, 30 °C, 37 °C and 50 °C for 5 min.
3. After incubation, add a few drops of iodine solution to each tube. Iodine turns starch blue; the lighter the colour, the more starch has been hydrolysed.
4. Record the colour intensity (visual chart or spectrophotometer at 620 nm). Calculate % starch hydrolysed.
5. Plot % activity against temperature to locate the optimum and illustrate the sharp fall at 50 °C (denaturation).

Suggested diagram

Summary checklist

• Enzymes are biological catalysts that are unchanged and can be reused.
• The active site has a unique three‑dimensional shape and specific functional groups.
• Specificity arises from complementary shape, charge and functional groups between enzyme and substrate.
• Lock‑and‑key = exact fit; induced‑fit = binding induces a conformational change that improves the fit.
• Only substrates that match the active site form an enzyme‑substrate complex and are converted to product.
• Temperature and pH give bell‑shaped activity curves; extremes cause denaturation.
• Competitive inhibitors block the active site; non‑competitive inhibitors alter enzyme shape.
• Genetic mutations can modify the active‑site shape and thus substrate specificity.

Practice questions

1. Explain why amylase cannot break down cellulose, even though both are polysaccharides of glucose.
2. Describe how a competitive inhibitor affects enzyme specificity and activity.
3. Give an example of an enzyme that follows the induced‑fit model and explain the structural change that occurs on substrate binding.
4. Design a simple experiment to determine the optimum temperature for an enzyme of your choice. Include the variables you would control and the observations you would record.
5. Briefly discuss how lactase deficiency leads to lactose intolerance and how this relates to enzyme specificity.