explain the mode of action of enzymes in terms of an active site, enzyme–substrate complex, lowering of activation energy and enzyme specificity, including the lock-and-key hypothesis and the induced-fit hypothesis

Mode of Action of Enzymes (Cambridge AS & A‑Level Biology 9700)

1. What is an Enzyme? (AO1)

• Enzymes are biological catalysts – usually globular proteins – that increase the rate of biochemical reactions without being consumed.
• They provide an alternative reaction pathway with a lower activation energy ( E_a ), allowing more reactant molecules to reach the transition state per unit time.

2. Enzyme Structure and the Active Site (Outcome 3.1.2)

• Overall structure: the three‑dimensional folding of the polypeptide chain creates a unique pocket or groove – the active site.
• Key amino‑acid side‑chains (examples):

  • Acidic residues (Asp, Glu) – act as proton donors/acceptors.
  • Basic residues (Lys, Arg, His) – stabilise negative charges or act as nucleophiles.
  • Ser, Cys, Tyr – provide nucleophilic –OH or –SH groups for covalent catalysis.
  • Hydrophobic residues – create a non‑polar micro‑environment that favours binding of hydrophobic substrates.

  These residues are directly involved in binding the substrate and in the chemical steps that convert substrate to product.

3. Enzyme–Substrate Complex (ES Complex)

• The substrate (S) binds to the active site of the enzyme (E) through non‑covalent forces (hydrogen bonds, ionic interactions, van der Waals forces, hydrophobic interactions).
• This temporary association forms the ES complex, the first step of the catalytic cycle.

4. Catalytic Cycle & Transition‑State Stabilisation

1. E + S → ES – substrate binds.
2. Induced fit – conformational change aligns catalytic residues and creates a favourable environment for the reaction.
3. Transition‑state stabilisation – the enzyme lowers the activation energy by stabilising the high‑energy transition state.
4. ES → EP – product(s) are formed while still bound.
5. EP → E + P – products are released; the enzyme returns to its original conformation.

5. Enzyme Specificity

Specificity is explained by two classic models.

5.1 Lock‑and‑Key Hypothesis (Emil Fischer, 1894)

• The active site is a rigid, pre‑formed cavity that exactly matches the shape of its substrate – like a key fitting into a lock.
• Explains why only substrates of the correct shape can bind.

5.2 Induced‑Fit Hypothesis (Daniel Koshland, 1958)

• The active site is flexible; substrate binding induces a conformational change that improves complementarity.
• The induced fit not only ensures specificity but also brings catalytic residues into optimal positions, enhancing the rate of reaction.

5.3 Comparison of the Two Models

6. Allosteric Regulation (Outcome 3.2.3 – optional but exam‑relevant)

• Allosteric enzymes have one or more regulatory (allosteric) sites separate from the active site.
• Binding of an effector molecule causes a conformational change that:

  • Positive (activator) – increases enzyme activity, usually by lowering K_m or raising V_max.
  • Negative (inhibitor) – decreases activity, often by raising K_m or lowering V_max.

• Typical example: phosphofructokinase‑1 in glycolysis, activated by ADP (positive) and inhibited by ATP (negative).

7. Factors Affecting Enzyme Activity (Outcome 3.1.3)

• Temperature – rate increases with kinetic energy up to an optimum; above this, denaturation reduces activity.

  • Denaturation – loss of native tertiary structure; can be reversible (heat‑shock) or irreversible (high heat, extreme pH, chemicals).

• pH – each enzyme has an optimum pH; extreme pH alters ionisation of active‑site residues and can cause denaturation.
• Enzyme concentration – more active sites → higher initial rate (V₀) until substrate becomes limiting.
• Substrate concentration – V₀ rises sharply at low [S] and approaches V_max as the enzyme becomes saturated.
• Co‑factors / prosthetic groups – non‑protein components required for activity (e.g., Mg²⁺ for DNA polymerase, heme in cytochrome c, NAD⁺ in dehydrogenases).
• Inhibitors

  Type	Binding site	Effect on Km	Effect on Vmax
  Competitive	Active site (competes with substrate)	↑ (apparent affinity ↓)	No change
  Non‑competitive	Allosteric site (binds whether or not substrate is bound)	No change	↓

8. Michaelis–Menten Kinetics (Outcome 3.2.2)

The relationship between initial rate (V₀) and substrate concentration ([S]) is described by:

\( V0 = \dfrac{V{\max}[S]}{K_m + [S]} \)

• V_max – maximum rate when all enzyme molecules are saturated with substrate.
• K_m – substrate concentration at which V₀ = ½ V_max; a measure of affinity (low K_m = high affinity).
• Competitive inhibitors increase the apparent K_m (more substrate needed) but do not affect V_max.
  

  Non‑competitive inhibitors lower V_max without changing K_m.

Data handling in the classroom

1. Measure V₀ at several substrate concentrations using a colourimetric assay (see Section 9).
2. Plot V₀ against [S] – the curve approaches a horizontal asymptote (V_max).
3. Linearise the data with a Lineweaver‑Burk plot (1/V₀ vs 1/[S]); y‑intercept = 1/V_max, x‑intercept = –1/K_m.
4. Calculate V_max and K_m and comment on the effect of any inhibitor added.

9. Practical Investigation of Enzyme Activity (Outcome 3.1.2)

Typical school‑level experiments use colourimetry to follow product formation or substrate depletion.

9.1 Catalase assay (H₂O₂ → H₂O + O₂)

• Measure the volume of O₂ produced in a fixed time (gas‑collection tube) or the decrease in H₂O₂ concentration using a spectrophotometer at 240 nm.
• Plot O₂ volume (or absorbance change) against time to obtain the initial rate (V₀).

9.2 Amylase assay (Starch → Maltose)

• Add iodine to the reaction mixture; iodine forms a blue‑black complex with starch.
• Loss of colour (decrease in absorbance at ~620 nm) indicates starch breakdown.
• Convert absorbance to concentration with a calibration curve (Beer–Lambert law) and plot concentration vs time.

Using a colourimeter

1. Prepare a blank containing all reagents except the enzyme.
2. Set the wavelength appropriate for the assay (e.g., 620 nm for iodine‑starch).
3. Record absorbance at regular intervals after adding the enzyme.
4. Convert absorbance to concentration, plot, and determine V₀ for each substrate concentration.

10. Immobilised Enzymes (Outcome 3.2.4)

• Definition – enzymes are fixed to a solid support (agarose beads, glass beads, polymer membranes) while retaining catalytic activity.
• Advantages

  • Easy separation of enzyme from the reaction mixture – essential for industrial processes.
  • Often more stable to temperature and pH extremes.
  • Reusable, reducing cost.

• Example – immobilised glucose oxidase on a membrane in blood‑glucose test strips.

11. Safety & Precision Tips for Enzyme Experiments

• Wear safety goggles and gloves when handling hydrogen peroxide, strong acids or bases.
• Always prepare a blank containing all reagents except the enzyme to correct for background absorbance.
• Start timing as soon as the enzyme is added and keep reaction times consistent.
• Use a calibrated colourimeter and record the temperature of each assay; temperature fluctuations affect rates.
• Label all tubes clearly and clean the cuvette between measurements to avoid cross‑contamination.

12. Key Points to Remember

• Enzymes are protein catalysts that lower activation energy by stabilising the transition state.
• The active site contains specific amino‑acid side‑chains that bind substrate and participate directly in the chemical reaction.
• Binding may follow the lock‑and‑key model (rigid fit) or the induced‑fit model (flexible fit); the latter better explains catalytic rate enhancement.
• Allosteric regulation allows effectors to modify V_max and/or K_m by changing enzyme conformation.
• Activity is influenced by temperature, pH, enzyme & substrate concentrations, co‑factors, denaturation, and inhibitors (competitive vs non‑competitive).
• Michaelis–Menten kinetics (V_max, K_m) quantify how rate depends on substrate concentration and how inhibitors alter these parameters.
• Practical colourimetric assays (catalase, amylase) enable students to generate data for kinetic analysis and to calculate V_max and K_m.
• Immobilised enzymes illustrate industrial applications and the concept of enzyme reuse.