The rate of an enzyme-catalyzed reaction is generally dependent on temperature. As temperature increases, the rate of the reaction typically increases up to a certain point. This is because higher temperatures provide more kinetic energy to the molecules, leading to more frequent collisions between the enzyme and substrate, and a greater proportion of collisions having sufficient energy to overcome the activation energy. This results in a higher frequency of enzyme-substrate complex formation and faster product formation.

However, beyond an optimal temperature, the rate of the reaction will decrease. This is because at high temperatures, the enzyme's tertiary and quaternary structures begin to break down (denature). Denaturation disrupts the enzyme's 3D conformation, including the shape of the active site. This loss of shape prevents the enzyme from binding to the substrate effectively, and the catalytic activity is lost. Therefore, the rate of the reaction decreases significantly as the temperature exceeds the optimal temperature.

The optimal temperature for an enzyme is the temperature at which the enzyme exhibits its maximum catalytic activity. This temperature varies depending on the enzyme and the organism from which it is derived. Enzymes from organisms living in colder environments typically have lower optimal temperatures than enzymes from organisms living in warmer environments. The relationship between temperature and enzyme activity can be represented graphically, with a bell-shaped curve showing the increasing rate up to the optimal temperature and then a sharp decline as the temperature rises above it.

Experimental Procedure:

1. Preparation: Prepare a solution of the enzyme and the substrate at a known concentration. Ensure the pH and temperature are kept constant throughout the experiment.
2. Reaction Setup: Set up the colorimeter with a fixed path length cuvette. Ensure the cuvette is filled with a blank solution (containing all components *except* the enzyme). This blank is used to zero the colorimeter.
3. Reaction Initiation: Add the enzyme to the blank solution in the cuvette. Start the timer immediately.
4. Data Collection: At regular time intervals (e.g., every 30 seconds, 1 minute), take absorbance readings from the colorimeter. Record the time and the corresponding absorbance value. Repeat the measurements multiple times (at least three replicates) to improve accuracy.
5. Control: Include a control experiment where the substrate is omitted. This will help identify any absorbance changes not due to the enzyme-catalysed reaction.

Variables to Consider:

• Substrate Concentration: Vary the initial substrate concentration systematically. Use a range of concentrations to observe the effect.
• Enzyme Concentration: Keep the enzyme concentration constant throughout the experiment.
• Temperature: Maintain a constant temperature throughout the experiment. Consider performing the experiment at different temperatures to investigate the effect of temperature on the reaction rate.
• pH: Maintain a constant pH throughout the experiment. Consider performing the experiment at different pH levels to investigate the effect of pH on the reaction rate.

Determining the Order of the Reaction:

The data obtained from the colorimeter (absorbance vs. time) can be used to determine the order of the reaction with respect to substrate concentration. The following approaches can be used:

• Initial Rate Method: Determine the initial rate of the reaction for different substrate concentrations. The initial rate is the slope of the absorbance vs. time curve at the very beginning of the reaction (when the substrate concentration is much higher than the enzyme concentration).
• Graphical Analysis: Plot the initial rate against the substrate concentration.
  • If the plot is linear, the reaction is first order with respect to the substrate.
  • If the plot is a straight line with a non-zero y-intercept, the reaction is zero order with respect to the substrate.
  • If the plot is a curve, the reaction is second order with respect to the substrate.

Method:

1. Catalase Preparation: Prepare a catalase extract by grinding fresh potato (or other suitable source) with distilled water. Filter the mixture through several layers of muslin to obtain a catalase solution. The concentration of the catalase solution should be kept constant for all trials.
2. Hydrogen Peroxide Solution: Prepare a solution of hydrogen peroxide (H₂O₂) of a known concentration (e.g., 3%).
3. Reaction Setup: Set up a system where the hydrogen peroxide solution is mixed with the catalase solution. A suitable apparatus would involve a flask containing the catalase solution, with a delivery tube leading to a graduated conical flask. The conical flask will collect the oxygen gas produced as a product.
4. Rate Measurement: Measure the volume of oxygen gas produced over a fixed time interval (e.g., 1 minute) for several trials. The volume of oxygen can be determined by measuring the displacement of water in the conical flask. Repeat the experiment at different temperatures (e.g., 10°C, 20°C, 30°C, 40°C) to investigate the effect of temperature on the reaction rate.
5. Control Variables: Crucially, maintain constant:

   • Catalase concentration
   • Hydrogen peroxide concentration
   • Volume of catalase solution
   • Temperature
   • Volume of hydrogen peroxide solution

Variables to Investigate: The independent variable is temperature. The dependent variable is the rate of oxygen production (volume of O₂ produced per minute).

Constant Conditions: Maintain constant temperature using a water bath. Ensure the same volume of hydrogen peroxide and catalase are used in each trial. Use the same type and concentration of catalase extract. Ensure the same apparatus is used for each trial.