Hypothesis: An enzyme immobilised in alginate will exhibit a lower rate of reaction compared to the same enzyme free in solution due to reduced accessibility of the active site to the substrate. However, the immobilised enzyme will retain activity over a longer period.

Methodology:

1. Enzyme Preparation: Obtain a purified enzyme (e.g., catalase from potato).
2. Immobilisation: Immobilise the enzyme in alginate beads using a suitable protocol (e.g., dropping enzyme solution into a calcium chloride solution). Ensure beads are of a consistent size.
3. Reaction Setup: Prepare two reaction setups: one with immobilised enzyme beads and one with free enzyme in a buffer solution. Maintain identical buffer conditions (pH, temperature).
4. Substrate Addition: Add a known concentration of substrate (e.g., hydrogen peroxide for catalase) to both reaction setups.
5. Measure Reaction Rate: Measure the rate of reaction over a set period (e.g., by measuring the volume of oxygen produced or the change in absorbance of a product). Repeat measurements multiple times for each setup.
6. Control Variables: Ensure consistent temperature, pH, substrate concentration, and enzyme concentration in both setups. Use the same volume of buffer.
7. Data Analysis: Calculate the reaction rate for each setup. Compare the rates using graphs and statistical analysis (e.g., mean and standard deviation).

Controlled Variables:

• Temperature: Maintain a constant temperature using a water bath.
• pH: Use a buffer solution to maintain a constant pH.
• Substrate Concentration: Use the same concentration of substrate in both setups.
• Enzyme Concentration: Use the same amount of enzyme in both setups.
• Volume of Buffer: Use the same volume of buffer in both setups.
• Bead Size (for immobilised enzyme): Ensure all alginate beads are of a similar size.

a) The concentration of Inhibitol is inversely proportional to the rate of oxygen production. As the concentration of Inhibitol increases, the rate of oxygen production decreases. This indicates that Inhibitol is an inhibitor of catalase activity.

b) The inhibition caused by Inhibitol is non-competitive. This is because the rate of oxygen production remains relatively constant at 25 µmol/min (the control) despite increasing concentrations of Inhibitol. In a competitive inhibitor, the rate would decrease proportionally to the Inhibitol concentration up to a certain point (when the enzyme becomes saturated with substrate). Since the rate remains relatively constant, Inhibitol must be binding to a site other than the active site, causing a conformational change that reduces the enzyme's catalytic efficiency.

c) Increasing the concentration of substrate (hydrogen peroxide) would not overcome the effect of the non-competitive inhibitor. In a non-competitive inhibitor, the inhibitor affects the enzyme's ability to catalyze the reaction, regardless of the substrate concentration. Therefore, even with a high concentration of substrate, the enzyme will not be able to achieve its maximum reaction rate (Vmax) because the inhibitor is still preventing the enzyme from functioning optimally.

a) Substrate Concentration: The rate of an enzyme-catalyzed reaction is directly proportional to the substrate concentration, up to a certain point. This is described by Michaelis-Menten kinetics. As the substrate concentration increases, more substrate molecules will collide with enzyme active sites, leading to a higher frequency of successful enzyme-substrate complex formation and thus a faster reaction rate. Eventually, the enzyme becomes saturated with substrate, meaning all active sites are occupied. Further increases in substrate concentration will not increase the reaction rate; the Vmax has been reached.

b) Enzyme Concentration: The rate of an enzyme-catalyzed reaction is directly proportional to the enzyme concentration, provided that there is sufficient substrate available. More enzyme molecules mean more active sites are available to bind substrate and catalyze the reaction, leading to a faster reaction rate. However, if the substrate concentration is limiting, increasing the enzyme concentration will not significantly increase the reaction rate.

c) Temperature: Increasing the temperature generally increases the rate of an enzyme-catalyzed reaction, up to a point. This is because higher temperatures increase the kinetic energy of the molecules, leading to more frequent and more energetic collisions between enzyme and substrate. However, at high temperatures, the enzyme can denature (lose its tertiary structure), which disrupts the active site and renders the enzyme inactive. Therefore, there is an optimal temperature for each enzyme, and reaction rates will decrease significantly above this temperature.

d) pH: The rate of an enzyme-catalyzed reaction is also affected by pH. Enzymes have an optimal pH at which they function most effectively. Changes in pH can alter the ionization state of amino acid residues in the enzyme's active site, which can affect substrate binding and catalytic activity. Extreme pH values (either very acidic or very alkaline) can denature the enzyme, leading to a loss of activity. The optimal pH varies depending on the enzyme and its environment.