Biology of Cell Lab (BIOL1021) Page 4 of 4 Lab 5 – Enzyme kinetics
EXPERIMENT OBJECTIVE:
The purpose of this experiment is to understand enzyme catalysis. Students will perform an enzyme assay and determine the rate of a biochemical reaction.
BACKGROUND INFORMATION
A catalyst is a substance that accelerates the rate of a chemical reaction without being consumed or transformed by the reaction. The catalyst does not alter the equilibrium constant of the reaction. Only the rate of approach to equilibrium is changed. A catalyst is not required in stoichiometric quantities and is often used in trace amounts. Platinum, palladium, strong acids, and bases are frequently used catalysts in organic chemistry and can accelerate reactions thousands of times.
The biological catalysts known as enzymes catalyze the great majority of chemical reactions in the cell. Enzymes can accelerate reactions 1014 to 1020 times, amounts that are far greater than any artificial catalyst. Enzymes generally produce these accelerations under the comparatively mild physiological conditions of neutral pH, atmospheric pressure and temperatures of 37°C. Unlike most catalysts; enzymes are generally very specific for the reactions they catalyze. The activity of certain enzymes can also be regulated by intracellular concentrations of key metabolites not directly involved with the reaction. This regulation can increase or decrease the activity of the enzyme in a manner adaptive to the cell’s physiological requirements at a given time. Enzymes that are regulated in this way are termed allosteric.
Certain proteins also contain, as integral parts of their structure, chemical groups that are not part of the amino acid residues but are absolutely required for biological activity. These groups include small organic molecules, such as certain vitamin derivatives, and certain metal ions. Groups such as these are called prosthetic groups. A well-known prosthetic group is heme. Heme consists of an iron atom coordinated to the nitrogens of a set of organic rings called porphyrin, giving a red color in highly concentrated solutions. The reactant molecule in an enzyme-catalyzed reaction is called the substrate. The substrate (S) is transformed to product (P). Before the enzyme can transform the substrate it must first bind to it. Initial binding is non-covalent and can be in rapid equilibrium. After productive binding has been achieved, the enzyme-substrate complex can now generate product that is subsequently released. The free enzyme (E) can now react with more molecules of substrate (the enzyme has turned over). This can be summarized using a single substrate, single product, and non-reversible reaction:
E + S Û ES ® EP ® E + P
If the reaction has 2 substrates, then there needs to be a S1 and S2 in the beginning part of the equation, also if there are 2 products this must be reflected at the end of the equation.
The disappearance of S or the appearance of P (or both) can be measured as a function of time. This relationship is the rate of the reaction. The method of measurement is called the assay. At a fixed enzyme concentration and fixed reaction conditions, increasing substrate concentrations can increase the reaction rate. The probability of forming more ES complex increases when there are more substrate molecules present. Generally, the substrate concentration is thousands of times greater than the enzyme concentration in kinetic studies performed in vitro. At the early stages of the reaction, if the substrate concentration is in great excess, the rate is approximately linear with time and is termed the initial velocity (v).
[S]1 - [S]2
T1 - T2
where [S]1 is the molar concentration of substrate at some initial time, T1, and [S]2 is the substrate concentration at a later time, T2. The reaction rate can also be expressed in terms of the appearance of product:
[P]2 - [P]1
T2 - T1
Note that the concentration of substrate decreases with time and the concentration of product increases with time. Graphically, this can be represented with the substrate concentration on the y-axis and time on the x-axis. The decrease in the substrate concentration with time will generate a curve. The rate of decrease is fastest at the earliest times of the reaction since the substrate concentration is comparatively high. The rate of decrease diminishes at later times because the substrate concentration is lower and the reaction is slower. Within small time intervals there will be sections of the curve that are approximately linear and the rate can be estimated. The rate of an enzyme reaction cannot be increased indefinitely by continuously increasing the substrate concentration. At some substrate concentration, all the enzyme molecules are bound to substrate and are involved in some stage of the catalytic cycle. Under these conditions the enzyme is saturated with substrate and no further increase in reaction velocity is observed. This does not mean that the enzyme is not functioning, it only means that the rate is at its maximum
Only a relatively small portion of the enzyme molecule is involved with substrate binding and catalysis. This region is called the active site. The active site contains the critical amino acid residues and, if applicable, the prosthetic groups required for activity.
Hydrogen peroxide is a toxic by-product of aerobic oxidation and certain processes in intermediary metabolism. All aerobic life forms have evolved methods of enzymatic peroxide detoxification. Almost all the cell types in mammals contain catalase, with liver, kidney and erythrocytes being particularly rich sources. The enzyme catalase (H2O2:H2O2 oxidoreductase) catalyzes the rapid decomposition of hydrogen peroxide by the following reaction:
2 H2O2 ———> 2H2O + O2 (gas)
Bovine liver catalase has a molecular weight of approximately 250,000 and is a tetramer of 4 identical subunits. The enzyme contains 4 heme prosthetic groups per molecule, one per subunit. The heme forms part of the active site and there are 4 active sites per molecule of enzyme. The peroxide oxygens are believed to be coordinated to the heme iron during one phase of the catalytic cycle. Another molecule of peroxide is then used to complete the reaction. Catalase has one of the highest turn over rates known. Over 3.6 x 107 molecules of hydrogen peroxide are decomposed per minute per molecule of enzyme.
We will use a colorimetric assay to measure the concentration of hydrogen peroxide based on the oxidation of iodide by peroxide:
2 I- + 2 H+ + H2O2 ———> 2 H2O + I2
The generation of iodine imparts a brown-red color to the solution. The color intensity increases with the peroxide concentration. Consequently, catalase mediated decomposition of hydrogen peroxide will decrease the color intensity of the assay with time. The stoichiometry of this reaction creates 1 mole of iodine for every mole of hydrogen peroxide. The assay has been designed to use a minimum of acid (less than 10 mM HCl). For a typical catalase assay the iodine concentration generated is less than 0.2 mM.
Student Experimental Procedures
The enzyme catalase catalyzes the decomposition of hydrogen peroxide (substrate) to water and oxygen gas (products). In this experiment:
• Catalase will be added to a buffered solution of hydrogen peroxide. A time course of the reaction will be obtained by removing aliquots from the reaction tube every 30 seconds.
• These aliquots will be added to separate tubes of assay solution. The assay solution denatures the enzyme, catalase, which destroys its activity.
• The iodide (I-) in the assay solution is oxidized by any remaining peroxide, producing a red-brown iodine (I2) solution. The color intensity can be quantitated in the spectrophotometer and the rate of the reaction determined.
• The concentrations of peroxide and enzyme in the reaction are approximately 1.8 milliMolar and 5 nanoMolar respectively.
Biology of Cell Lab (BIOL1021) Page 4 of 4 Lab 5 – Enzyme kinetics
Biology of Cell Lab (BIOL1021) Page 4 of 4 Lab 5 – Enzyme kinetics
The class will perform this lab in groups of three. Each group will perform the reactions at either 4°, RT or 37°. All the results from each groups assay will be placed on the board. It is your responsibility to get the data written down. You will calculate the average absorbance obtained for each time point at each temperature. Your homework will be to perform the analysis described at the end of the lab sheets and answer the study questions. This will be turned in at the start of the next lab. Please answer questions on a separate sheet of paper.
PREPARATION OF ASSAY TUBES
1. With a water resistant pen, label 6 empty test tubes (at the top) to indicate blank and the various reaction times (0 to 2 minutes): B (blank)
0
0.5
1.0
1.5
2.0
2. With a 5 ml pipet, transfer 3 ml of assay solution to each of the tubes. Set pipet aside for disposal.
3. With a 1 ml pipetor with a tip, transfer 0.3 ml (300 ml) of diluted buffer to the blank. Mix by tapping. Discard tip.
PREPARATION OF CONTROL AND REACTION TUBES
4. Label one of two remaining test tubes "Con" (for control) and the other "Rxn" (for reaction).
5. With a 5 ml pipet, dispense 1.8 ml of Enzyme Reaction Cocktail to each of the two tubes.
6. With a 1 ml pipetor with tip, add 0.3 ml (300ml) of dilute phosphate buffer to the control tube (Con). Discard pipet tip.
7. Remove 0.3 ml of liquid from the control tube (Con) and add to the assay tube labeled 0. Set the "Con" tube aside as this is your negative control. Discard pipet tip.
8. The remaining steps will be conducted using the Rxn tube. Make sure you are prepared for the following steps as once you add the enzyme, you must start collecting samples every 30 seconds.
Biology of Cell Lab (BIOL1021) Page 4 of 4 Lab 5 – Enzyme kinetics
Enzyme reaction cocktail is added. Hydrogen peroxide that is not catalyzed by the enzyme catalase will oxidize iodide to give a brown-red color. The color intensity increases with the peroxide concentration. Upon addition of enzyme catalase, the reaction will begin. Start timing the reaction immediately and aliquot 0.3 ml of this mixture to tubes labeled 0.5, 1.0, 1.5 and 2.0 at 30 second intervals. Do NOT start this step until you are really ready. Make sure you understand each of the following steps.
Biology of Cell Lab (BIOL1021) Page 4 of 4 Lab 5 – Enzyme kinetics
Preparation and Monitoring of Timed Reactions
9. With a 1 ml pipetor with tip, add 0.3 ml of diluted catalase (Enzyme) to the tube you labeled Rxn. Mix. Start timer, or note the second hand on the clock or watch. Discard pipet tip and immediately get a new one to take samples.
10. With the 1 ml pipet, remove 0.3 ml from the Rxn tube and at 0.5 min. (30 seconds), add it to tube 0.5. Mix.
11. With the 1 ml pipet, remove 0.3 ml from the Rxn tube and at 1 minute, add it to tube 1. Mix.
12. With 1 ml pipet, remove 0.3 ml from the Rxn tube and at 1 minute 30 seconds, add it to tube 1.5. Mix.
13. With 1 ml pipet, remove 0.3 ml from the Rxn tube and at 2 minutes, add it to tube 2.0. Mix. Discard pipet tip.
14. Wait 4 minutes after your last time point to allow full color development.
DATA COLLECTION
Spectral readings can now be taken. You may insert your test tubes directly into the instrument. Wipe the outside of each tube to remove any dust or fingerprints that may interfere with the reading.
15. Zero the instrument with the tube B solution (Blank) according to your instructor’s directions. Be sure the instrument is set at 500 nm wavelength. The instrument should read 0 absorbance with the blank solution (no color).
16. Remove the blank and record the absorbance values for each solution in tubes 0 to 2.0. Record the results in the table. If you are using an instrument such as a Spec 20, it should take approximately two to three minutes to complete your readings.
Time (Min) / Assay Solution / Diluted Buffer / Volume Con / Volume Rxn / A500Blank / 3 ml / 0.3 ml / --- / ---
0 / 3 ml / --- / 0.3 ml / ---
0.5 / 3 ml / --- / --- / 0.3 ml
1.0 / 3 ml / --- / --- / 0.3 ml
1.5 / 3 ml / --- / --- / 0.3 ml
2.0 / 3 ml / --- / --- / 0.3 ml
Analysis
The reaction rate can be obtained by graphing the absorbance data versus time. However, the rate can also be expressed in terms of substrate consumed.
1. To express your data in terms of molar concentration of peroxide:
Absorbance x 11 = Molarity of hydrogen peroxide in Rxn tube.
e
e is the extinction coefficient for this assay system and has been determined by your instructor and is 3.7 x 103. Multiplication by 11 (dilution factor) gives the peroxide concentration in the reaction tube. Scientific notation will make the calculations more convenient.
2. Get all the data from the board that was done by all of the groups in the lab. For each temperature, calculate the average absorbance for each time point. In case you forgot: Average is the sum of all observations at 1 time point divided by the number of observations. You should end up with 3 sets of numbers to graph on the graph paper.
3. Graph the peroxide concentration on the y-axis versus time on the x-axis.
4. Draw the best straight line through the data points. You may notice some curvature to the data points. This is normal, especially between 0 and the first time point, and between later time points. You are making a linear approximation.