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

Learning Objective

Investigate the progress of enzyme‑catalysed reactions by:

• Measuring the rate of formation of products using catalase.
• Measuring the rate of disappearance of substrate using amylase.

1. Key Concepts

Enzymes are biological catalysts that speed up reactions by lowering the activation energy (\$E_a\$). They bind substrates at the active site, forming an enzyme–substrate complex (ES) which then converts to product(s) and regenerates the free enzyme.

2. General Rate Equation

The instantaneous rate of an enzyme‑catalysed reaction can be expressed as:

\$\$

v = \frac{d[P]}{dt} = \frac{V{max}[S]}{Km + [S]}

\$\$

where \$[P]\$ is product concentration, \$[S]\$ is substrate concentration, \$V{max}\$ is the maximum rate, and \$Km\$ is the Michaelis constant.

3. Experimental Investigation

3.1 Catalase – Measuring Product Formation

Catalase catalyses the decomposition of hydrogen peroxide (\$\mathrm{H2O2}\$) into water and oxygen:

\$\$

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

\$\$

3.1.1 Apparatus

3.1.2 Procedure (Outline)

1. Prepare a series of test tubes each containing 5 mL of \$\mathrm{H2O2}\$.
2. Add a fixed volume (e.g., 0.5 mL) of enzyme source to each tube and start the stopwatch immediately.
3. Collect the volume of \$\mathrm{O_2}\$ released at regular intervals (e.g., every 10 s) using the gas syringe.
4. Repeat the experiment at different temperatures (e.g., 10 °C, 25 °C, 37 °C, 50 °C) to study temperature effects.
5. Perform a control tube with no enzyme to account for any non‑enzymatic decomposition.

3.1.3 Data Recording

3.1.4 Calculations

Rate of product formation (\$v\$) can be obtained from the slope of the volume‑vs‑time graph:

\$\$

v = \frac{\Delta V{\mathrm{O2}}}{\Delta t}

\$\$

Convert volume to moles using the ideal gas equation (\$PV = nRT\$) if required.

3.2 Amylase – Measuring Substrate Disappearance

Amylase hydrolyses starch into maltose and glucose:

\$\$

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

\$\$

3.2.1 Apparatus

3.2.2 Procedure (Outline)

1. Add 5 mL of starch solution to each test tube.
2. Introduce a fixed volume (e.g., 0.5 mL) of amylase source and start timing.
3. At predetermined intervals (e.g., every 30 s), withdraw 0.5 mL of reaction mixture and add it to 2 mL of iodine solution.
4. Observe the colour change: deep blue‑black indicates starch still present; colourless indicates complete hydrolysis.
5. Record the time at which the colour disappears (or absorbance falls below a set threshold).
6. Repeat at different pH values (using appropriate buffers) to study pH effects.

3.2.3 Data Recording

3.2.4 Calculations

Convert absorbance to concentration of starch using Beer‑Lambert law:

\$\$

A = \varepsilon \, l \, [\text{Starch}]

\$\$

Rate of substrate disappearance (\$v_{-S}\$) is then:

\$\$

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

\$\$

4. Data Analysis and Interpretation

1. Plot volume of \$\mathrm{O2}\$ (or moles) against time for catalase; the initial linear portion gives \$v{+P}\$.
2. Plot starch concentration (or absorbance) against time for amylase; the slope of the linear early phase gives \$v_{-S}\$.
3. Determine \$V{max}\$ and \$Km\$ by varying substrate concentration and fitting data to the Michaelis–Menten equation (use Lineweaver–Burk double‑reciprocal plot if required).
4. Compare the effect of temperature and pH on the two enzymes; identify optimum conditions and discuss denaturation.

5. Sources of Error and Improvements

• Inaccurate timing – use electronic timers.
• Leakage of gas in the catalase set‑up – ensure airtight connections.
• Incomplete mixing of reagents – vortex briefly after each addition.
• Variability in enzyme concentration – standardise protein content using a Bradford assay.
• Temperature fluctuations – use a calibrated water bath with ±0.5 °C stability.

6. Summary

Enzyme kinetics can be explored by measuring either the appearance of product (catalase) or the disappearance of substrate (amylase). Both approaches rely on accurate, time‑resolved data collection and allow calculation of reaction rates, \$V{max}\$, and \$Km\$. Understanding how temperature and pH affect these rates provides insight into enzyme structure–function relationships, a key component of the Cambridge A‑Level Biology curriculum.