shown by line A. The net effect of temperature on an enzyme-catalyzed reaction is given by line C.

Transcription

1 Experiment 8 EFFECT OF TEMPERATURE ON ENZYME ACTIVITY Temperature affects the stability of an enzyme as well as the binding of substrate and its transformation to product. Line B of Fig. 8-l shows the effect of temperature on the stability of the enzyme. At lower temperatures the enzyme is stable; however, the velocity of denaturation becomes faster the higher the temperature. Consequently, less active enzyme is available for conversion of substrate to product at the higher temperatures. Line A of Fig. 8-l shows the effect of temperature on the velocity of conversion of substrate to product. For enzyme-catalyzed reactions the velocity will increase from 1.4 to 3 times for each 10 C rise in temperature. (At E a = 6,000, 12,000 and 18,000 cal/mol, the factors are 1.4, 2.0 and 3.0, respectively). Theoretically the velocity should increase at an ever-accelerating rate as the temperature is increased as shown in line A. The net result of increasing temperature on increasing the rate of conversion of substrate to product and on increasing the rate of denaturation of the enzyme is line C of Fig Figure 8-1. Effect of temperature on the velocity of an enzyme-catalyzed reaction. The right hand y-axis is for line B which shows the rate of denaturation of enzyme as a function of temperature. Effect of temperature on conversion of substrate to product is 1

2 shown by line A. The net effect of temperature on an enzyme-catalyzed reaction is given by line C. The so-called "temperature optimum" of the enzyme is at the maximum of line C. The "temperature optimum" is not a unique characteristic of an enzyme; rather, it applies to the entire system since ph, ionic strength, dielectric constant, substrate concentration, etc. often affect the observed optimum as well as the incubation or reaction time employed. The data of line C of Fig. 8-1 are plotted as 2.3 log k versus 1/T(K) in Fig. 8-2 according to the Arrhenius relationship, k = Ae-E a /RT. The positive slope on the left side gives a measure of activation energy, E a, for denaturation of the enzyme. E a for denaturation is usually in the range of 35,000 to 175,000 cal/mol. Thus, a few degrees change in temperature above the "temperature optimum" has a tremendous effect on rate of denaturation. This is why an enzyme may be stable at a given temperature but quite unstable at a temperature just 4-5 higher. Figure 8-2. Effect of temperature on an enzyme-catalyzed reaction as plotted according to the Arrhenius relationship, k = Ae-E a /RT. 2

3 The negative slope on the right side of Fig. 8-2 gives a measure of E a for conversion of substrate to product. If [S]o >> K m then E a will be for the ratedetermining step in the reaction. E a for conversion of substrate to product is usually in the range of 6,000 to 12,000 cal/mol but may occasionally have a value as high as 18,000 cal/mol. REAGENTS: Milk or bovine intestine alkaline phosphatase, as described in Experiment 7. Buffer. 0.2 M glycine, ph Different buffers are prepared so as to have a ph of 9.75 at each temperature. Choose the correct one for each temperature. Substrate. 2.5 x 10-3 M disodium p-nitrophenyl phosphate. PROCEDURE: The water baths used to maintain the cell compartments of the spectrophotometers at a constant temperature will be set near 25, 30, 35, 40 and 45 C. Please do not change these settings. For this experiment it will be necessary to use the Shimadzu and the Bausch and Lomb Spectronic 20 spectrophotometers. Make sure you read Appendix F to become familiar with the operation of the latter. When doing your experiment read the thermometer and record the exact temperature to the nearest 0.1. The reactions, to be done in duplicate at each temperature, will consist of 2.0 ml of 0.2 M glycine buffer, ph 9.75, at the temperature being used, 2.0 ml of 2.5 x 10-3 M substrate, 0.80 ml of deionized water and 0.20 ml of enzyme. All reagents except enzyme are to be placed in a test tube or spectrophotometer tube and equilibrated for a minimum of 5 min at the temperature being used. About 0.25 ml of enzyme in a test tube should be placed in the water bath and equilibrated not more than 30s. Always equilibrate a new aliquot of stock enzyme before each determination. Following equilibration, add 0.20 ml enzyme to the reaction tube, mix and rapidly return the tube to the spectrophotometer. Record absorbance at 5 sec intervals for 3 min. QUESTIONS: 1. From your value for the activation energy, calculate the time of enzyme assay at 0 C that would produce the same amount of product as 1 min at 30 C. 2. Was the enzyme stable at 45 C? Give reasons for your answer. 3

4 3. What percentage error would there be in determining the rate of this enzymecatalyzed reaction if the temperature, supposedly 40 C, was actually 38 C? 4. The Arrhenius relationship, k = Ae-E a /RT indicates that the slope of the line is -E a /R when 2.3 log k versus 1/T (K) is plotted. What mistake is made in your determination of E a when 2.3 log initial velocity versus 1/T is plotted for a reaction where [S] >> K m? Where [S] << K m? 4

5 EXPERIMENT 8 Effect of Temperature on Enzyme Activity Laboratory Report Name: Date: 1. Submit a graph of absorbance change versus time at each temperature. 2. Determine the initial velocity (moles/liter/min) of each individual reaction and present in table form along with measured temperature. 3. Submit a graph of log initial velocity versus 1/T. 4. Calculate activation energy for the reaction. 5. Submit answers to questions. 5