Enzyme Catalysis

Overview In this laboratory you will use computer interfaced colorimeters to measure and then calculate the rate of an enzymatic controlled reaction.

Objectives Before doing this laboratory you should understand:  The general functions and activities of enzymes.  The relationship between the structure and function of enzymes.  The concept of initial reaction rates of enzymes.  How the concept of free energy relates to enzyme activity.  That changes in temperature, pH, enzyme concentration and substrate concentration can affect the initial reaction rates of enzyme-catalyzed reactions. After doing this laboratory you should be able to:  Measure the effects of changes in temperature, pH, enzyme concentration, and substrate concentration on reaction rates of an enzyme-catalyzed reaction in a controlled experiment.  Explain how environmental factors affect the rate of enzyme-catalyzed reactions.

Background The enzyme Polyphenoloxidase is found in many plant organs, which store food. Examples include potatoes, bananas, and apples. When the organ is cut or damaged by bruising, the enzyme is released into the cytoplasm. The enzyme causes a chemical reaction to take place that produces a brownish material called a Quinone. Quinones are bacterial inhibitors, which may protect the surrounding tissues from infection. In this laboratory exercise, polyphenoloxidase will be isolated from potatoes. A substrate called Catechol will be mixed with the enzyme. The rate of the enzymatic reaction will be followed by measuring the amount of colored quinone that is produced from the colorless solution of catechol. Factors that affect the enzyme such as isomers, pH, and substrate concentration will be studied.

Enzyme Preparation and Experimental Design Procedure A class set of the enzyme extract will be prepared. Peel several Irish potatoes and slice them into a blender. Add a small quantity of water (50-100 ml) and liquefy. The resulting pulp will be filtered through cheesecloth. The extract will be filtered a second time through filter paper. Approximately 2-5 ml of extract will be needed for each student group. The instructor may prepare the enzyme extracts in advance. Substrate and Isomer solutions Prepare .01 M solutions of catechol (substrate), hydroquinone (isomer) and resorcinol (isomer). The isomers have the same formula weights as the catechol, but differ in the position of the -OH groups on the carbon ring (structural isomers) as can be seen in the diagrams below:

Colorimeters The colorimeter probe is an instrument that uses a beam of light to measure transmittance and/or absorbance of solutions. The light is sent through a moveable grating, allowing specific wavelengths of light to be selected and passed through the sample. A phototube on the other side of the cuvette measures the amount of light that has passed through the sample and produces an electrical signal. The strength of the signal can be directly observed on the meter or can be sent to an interfaced computer. In this experiment, the starting solutions (with substrate) will be clear, allowing nearly 100% of the light to be transmitted through the sample tube. If the enzyme produces the product, the sample will darken, permitting less light to be transmitted through the cuvette. The change in transmittance will be directly proportional to the amount of product or quinone formed by the enzymatic reaction. If the transmittance readings are recorded over time, the rate of the enzymatic reaction can be calculated.

A “blank” tube will be used to cancel out the color of the enzyme extract. Therefore, any color changes observed by the spectrophotometer will be due to the conversion of substrate to product.

Note – If the amount of enzyme is changed from a test run as compared to the blank, the spectrophotometer readings will be slightly off. Therefore, this lab will focus on the curves (changes over time) produced during an experimental run, not the actual readings of the colorimeter.

Preparing the Computer for Data Collection Each run in this experiment should last 5 minutes. The timing feature should be set in the experimental set-up so that a reading is recorded every 5 seconds. The Y axis can be set for values of 0 to 100. If the data should run off the graph during a test run, remember that the "auto scale" icon can be used to rescale the graph after the experiment has been completed.

Reaction Mixtures The total volume in the cuvette in all tests will be 3.5 ml. Follow the directions given below for the amount of substrate or isomer to add to the cuvette. Add enough distilled water to bring the volume up to 3.5 ml. When the reaction mixture is ready, add 3-5 drops of the enzyme extract, mix quickly by inverting the cuvette, and place in the colorimeter as fast as possible. Once the cuvette is in place, data collection may begin. (See the Comment on the next page)

Treatments All students will run the positive control (the catechol alone treatment) and the negative control (no enzyme treatment). In addition, each team of students will choose one variable to manipulate and compare to the control as to reactivity. A class data set of all the reactions will then be available. The directions for preparing the positive and negative control treatments follow: 1. Catechol alone Treatment - the positive control (3 ml catechol + 3-5 drops enzyme)

2. No enzyme treatment- the negative control (3 ml catechol + 3-5 drops water)

Comment - The reaction will begin immediately with the addition of the enzyme. The computer must be preset to start recording the data before the enzyme extract is added to the solution. After the enzyme is added to the mixture, the cuvette should quickly inverted (gently to prevent excessive air bubbles which will lower transmittance) and placed in the colorimeter. Then, start recording the data immediately.

Possible variables: Each group must complete at least 2 additional runs. pH- either lower (acidic) or higher (basic) temperature- either colder or warmer (heat or cool the substrate not the enzyme) concentration of substrate (catechol) concentration of enzyme isomers- resorcinol or hydroquinone

Each group will be creating a mini poster to present in a poster session on Monday 9/18. Separate handouts will be provided with information on poster set up with examples. Enzyme reaction graphs and the following data calculations must be included on each poster.

Data Calculations In order to compare how enzyme activity was affected by the different situations, the rate of enzyme activity must be calculated. This can be done by using the following formula (basically it’s the slope!): Rate = Conc.2 - Conc.1 Time 2 - Time 1

In the first minute of an enzymatic reaction, the number of substrate molecules is usually so large compared to the number of enzyme molecules that changing the substrate concentration does not affect the number of successful collisions between substrate and enzyme. During this initial period, the enzyme is acting on substrate molecules at a more or less constant rate. The rate during this early period is called the Initial Velocity of the reaction. The rate of the enzyme activity will decrease after the initial velocity as the substrate is used up and the enzyme has fewer substrate molecules to act on. Calculate the Initial Velocity for each test run using the concentration values at time 0 and time 60 seconds. This will allow the initial velocity to be calculated on a per minute basis.