state that enzymes are globular proteins that catalyse reactions inside cells (intracellular enzymes) or are secreted to catalyse reactions outside cells (extracellular enzymes)

Mode of Action of Enzymes

Learning Objective

State that enzymes are globular proteins that catalyse reactions inside cells (intracellular enzymes) or are secreted to catalyse reactions outside cells (extracellular enzymes).

1. Definition & Classification

• Enzyme: a biological catalyst that is a globular protein (or a protein‑cofactor complex).
• Can act intracellularly (e.g., hexokinase, DNA polymerase) or be secreted to act extracellularly (e.g., amylase, pepsin).
• Many enzymes need a non‑protein partner – a cofactor (inorganic ion) or coenzyme (organic molecule) – for full activity.

2. Mode of Action

2.1 Active site and enzyme–substrate (ES) complex

Enzymes bind substrates at a highly specific region called the active site. The binding forms an enzyme–substrate complex (ES) that stabilises the transition state, thereby lowering the activation energy (Eₐ) of the reaction:

E + S ⇌ ES → E + P

2.2 Lock‑and‑key vs. Induced‑fit

• Lock‑and‑key model: the active site is a rigid “lock” that matches the substrate “key” exactly.
• Induced‑fit model: substrate binding induces a conformational change in the enzyme, improving complementarity and further lowering Eₐ.

2.3 Michaelis–Menten Kinetics (AO2)

The rate of an enzyme‑catalysed reaction follows the Michaelis–Menten equation:

v = \(\dfrac{V{\text{max}}[S]}{Km + [S]}\)

• V_max: maximum rate when every enzyme molecule is saturated with substrate.
• K_m: substrate concentration at which the reaction rate is half of V_max; a measure of enzyme affinity (low K_m = high affinity).

A Michaelis–Menten curve and its double‑reciprocal (Lineweaver‑Burk) plot are useful for interpreting kinetic data in exams.

3. Factors Influencing Enzyme Activity

4. Intracellular vs. Extracellular Enzymes

5. Immobilised Enzymes

• Enzymes can be trapped in a matrix (e.g., calcium‑alginate beads) to form an immobilised enzyme system.
• Advantages: easy recovery, reuse, continuous operation, greater stability against temperature/pH changes.
• Industrial example: immobilised glucose‑isomerase for high‑fructose corn syrup production.

6. Practical Applications (Cambridge AS/A‑Level)

6.1 Colourimetric Enzyme Assays

• Activity is measured by the change in colour of a product or substrate that absorbs at a known wavelength.
• Amylase assay: starch → maltose; the iodine‑starch complex (blue) disappears as starch is hydrolysed. The rate of colour loss (absorbance decrease) gives the reaction rate.
• Data are plotted as absorbance vs. time; the initial slope provides v₀ for kinetic analysis.

6.2 Investigation of Catalase and Amylase

1. Choose the enzyme: catalase (breaks down H₂O₂) or amylase (hydrolyses starch).
2. Vary one factor at a time – temperature, pH, substrate concentration, or inhibitor concentration.
3. Measure the rate:

   • Catalase – collect the volume of O₂ gas evolved (gas‑evolution syringe) or use a colourimetric H₂O₂ assay.
   • Amylase – monitor the disappearance of the blue iodine‑starch colour or use a DNS (3,5‑dinitrosalicylic acid) assay for maltose.

4. Plot the results (e.g., rate vs. temperature) to locate the optimum condition and calculate V_max and K_m from a Lineweaver‑Burk plot.
5. Discuss sources of error (temperature drift, inaccurate pipetting, incomplete mixing) and suggest ways to minimise them (water‑bath control, calibrated pipettes, replicates).

6.3 Suggested Investigation Design (General)

1. Prepare a series of substrate solutions of known concentrations.
2. Add a fixed amount of enzyme to each tube and start timing.
3. Record the change in absorbance (or gas volume) at regular intervals.
4. Determine the initial rate (v₀) for each substrate concentration.
5. Construct a Michaelis–Menten curve and a Lineweaver‑Burk plot to obtain V_max and K_m.
6. Repeat the experiment under different temperature or pH conditions to explore factor effects.

7. Suggested Diagram