investigate the progress of enzyme-catalysed reactions by measuring rates of formation of products using catalase and rates of disappearance of substrate using amylase

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

Learning Objectives

• Explain how enzymes accelerate reactions (active site, transition state, lock‑and‑key & induced‑fit, regeneration of the enzyme).
• Derive the Michaelis–Menten equation and calculate V_max, K_m and k_cat.
• Analyse the effect of temperature, pH, enzyme concentration, substrate concentration and inhibitors on enzyme activity.
• Distinguish reversible from irreversible inhibition and discuss the advantages and disadvantages of enzyme immobilisation.
• Design, carry out and evaluate practical investigations:

  • Measuring product formation with catalase.
  • Measuring substrate disappearance with amylase.

• Interpret kinetic data using linear‑reciprocal (Lineweaver–Burk) plots and recognise sources of error; be aware of alternative linearisations.

1. Enzyme Fundamentals

1.1 What is an Enzyme?

• Biological catalyst – lowers the activation energy (E_a) without being consumed in the overall reaction.
• Usually a protein (or a ribozyme) that adopts a specific three‑dimensional conformation.

1.2 Active Site and Transition State

• Active site: a small pocket containing residues that bind substrate(s) and stabilise the transition state.
• Binding brings substrates into the correct orientation and reduces the energy required to reach the transition state, thereby increasing the rate of product formation.
• Key interactions: hydrogen bonds, ionic bonds, hydrophobic contacts and, in some enzymes, a catalytic triad (e.g. Ser‑His‑Asp in serine proteases).
• After product release the enzyme returns to its original form – it is regenerated unchanged and can catalyse further cycles.

1.3 Models of Substrate Binding

1.4 Michaelis–Menten Kinetics

For a simple reversible reaction:

\[

E + S \;\underset{k{-1}}{\stackrel{k{1}}{\rightleftharpoons}}\; ES \;\underset{k{-2}}{\stackrel{k{2}}{\rightleftharpoons}}\; E + P

\]

Assuming the steady‑state (rate of formation of ES = rate of its breakdown) gives the Michaelis–Menten equation:

\[

v = \frac{V{\max}[S]}{K{m} + [S]}

\]

• V_max = k_cat[E]_total – maximum rate when every enzyme molecule is saturated with substrate.
• K_m = (k_-1 + k₂) / k₁ – substrate concentration at which the reaction rate is half of V_max.
• k_cat (turnover number) – number of substrate molecules converted per enzyme molecule per second.
• Specific activity – units of activity per milligram of protein; useful when the enzyme preparation is not pure.

1.5 Graphical Determination of Kinetic Parameters

• Hyperbolic plot of v versus [S] (Michaelis–Menten curve).
• Lineweaver–Burk plot (double‑reciprocal):

  \[

  \frac{1}{v} = \frac{K{m}}{V{\max}}\frac{1}{[S]} + \frac{1}{V_{\max}}

  \]

  – y‑intercept = 1/V_max, slope = K_m/V_max.

• Limitations: gives excessive weight to low‑[S] points and magnifies experimental error.
• Alternative linearisations (less error‑prone):

  • Eadie‑Hofstee: \(v = -K{m}\,(v/[S]) + V{\max}\)
  • Hanes‑Woolf: \([S]/v = [S]/V{\max} + K{m}/V_{\max}\)

  – Mention these when discussing why the double‑reciprocal method is discouraged for precise work.

2. Factors Affecting Enzyme Activity (Syllabus 3.2)

2.1 Reversible vs. Irreversible Inhibition

3. Practical Investigations

3.1 Catalase – Measuring Product Formation (O₂)

3.1.1 Principle

Catalase catalyses the rapid decomposition of hydrogen peroxide:

\[

2\;\mathrm{H2O2}\;\xrightarrow{\text{catalase}}\;2\;\mathrm{H2O}\;+\;\mathrm{O2}\uparrow

\]

3.1.2 Apparatus

3.1.3 Procedure (outline)

1. Label tubes for the required temperatures (e.g., 10 °C, 25 °C, 37 °C, 50 °C) and a control containing no enzyme.
2. Add 5 mL of 3 % H₂O₂ to each tube and equilibrate in the water bath for 2 min.
3. Insert the gas‑collection syringe, add 0.5 mL of the enzyme source, seal immediately and start the stopwatch.
4. Record the volume of O₂ at 10 s intervals for 1 min (or until the reaction ceases).
5. Repeat each temperature at least three times for reproducibility.

3.1.4 Data Recording

3.1.5 Calculations & Analysis

• Calculate the initial rate V (mL s⁻¹) from the slope of the early linear segment (usually the first 30 s).
• If a quantitative rate in moles s⁻¹ is required, convert volume to moles of O₂ using the ideal‑gas equation \(PV = nRT\) (standard temperature and pressure assumed).
• Plot temperature versus initial rate to identify the optimum temperature and discuss the decline at higher temperatures as a result of enzyme denaturation.

3.2 Amylase – Measuring Substrate Disappearance (Starch)

3.2.1 Principle

Amylase hydrolyses starch into maltose and glucose:

\[

\text{Starch} + \text{H}_2\text{O} \xrightarrow{\text{amylase}} \text{Maltose} + \text{Glucose}

\]

Residual starch reacts with iodine to give a blue‑black complex; loss of colour indicates substrate consumption.

3.2.2 Apparatus

3.2.3 Procedure (outline)

1. Prepare reaction mixtures containing 5 mL of starch solution and the appropriate buffer; equilibrate at the chosen temperature.
2. Add 0.5 mL of the amylase source, mix quickly, and start the stopwatch.
3. At 30 s intervals withdraw 0.5 mL of the reaction mixture and add it to 2 mL of iodine solution in a cuvette.
4. Record the colour change (qualitative) or measure absorbance at 620 nm (quantitative).
5. Continue until the colour disappears (A ≈ 0) or for a fixed total time (e.g., 3 min).
6. Repeat for each pH and temperature, performing at least three replicates per condition.

3.2.4 Data Recording (spectrophotometric example)

3.2.5 Calculations & Analysis

• Convert absorbance to starch concentration using Beer‑Lambert law \(A = \varepsilon\,l\,[\text{Starch}]\) (ε obtained from a calibration curve).
• Determine the initial rate of substrate disappearance:

  \[

  v_{-S} = -\frac{\Delta[\text{Starch}]}{\Delta t}

  \]

  using the linear portion of the early time‑course (e.g., 0–60 s).

• Plot initial rate against substrate concentration for different pH/temperature values to obtain Michaelis–Menten curves; if required, construct Lineweaver–Burk (or alternative) plots to extract V_max and K_m.

4. Data Analysis & Interpretation

1. Initial rates – draw v versus time; the slope of the early linear region gives the initial rate.
2. Temperature effect – plot temperature against initial rate; identify the optimum temperature and discuss loss of activity at higher temperatures as denaturation of the enzyme’s secondary/tertiary structure.
3. pH effect – present a pH‑rate profile; explain the optimum in terms of ionisation of active‑site residues.
4. Substrate‑concentration study – plot v against [S]; fit the data to the Michaelis–Menten equation (non‑linear regression) or use a Lineweaver–Burk plot to obtain V_max and K_m. Discuss the meaning of each parameter.
5. Enzyme‑concentration study – keep [S] ≫ K_m and vary enzyme amount; expect a linear relationship (v ∝ [E]), confirming that rate depends on the number of active sites available.
6. Inhibition study (optional) – add a known inhibitor (e.g., acarbose for amylase). Use Lineweaver–Burk plots to distinguish competitive, non‑competitive or uncompetitive inhibition.
7. Sources of error – timing inaccuracies, temperature fluctuations, incomplete mixing, gas‑loss in the catalase experiment, pipetting errors, and the intrinsic error amplification in double‑reciprocal plots. Suggest improvements such as using a digital data‑logger, pre‑warming reagents, and employing alternative linearisation methods.