Assignment 1

When you set up an experiment in EnzymeLab, you will add a buffered solution, fructose as substrate, invertase, and, in some reactions, inhibitors to a test tube to measure the rate of invertase activity. You will have the choice of performing each reaction at different temperatures and under different buffer conditions in which you can examine the effect of these parameters on invertase activity. A visible light spectrophotometer will measure product formation by measuring the absorbance of glucose (released as fructose is cleaved by invertase) at approximately 450 nm. Data are recorded and plotted as a function of product concentration [P] in micromoles (mm) versus time (minutes). Raw data you collect can then be analyzed by several different types of plots that are commonly used for analyzing kinetic data for enzyme-catalyzed reactions.


1 .		Develop a hypothesis to predict the effect of an increase in temperature on invertase activity, and enter it below. Then, working in EnzymeLab test your hypothesis.

2 .		After each time you add enzyme, a plot of product concentration versus time will appear with enzyme kinetic data plotted as data points in solid black circles. What did you observe for the plot of product concentration versus time? Is this what you expected? Explain your answer below.

3 .		What is the optimal temperature for invertase activity? Is this what you expected? Would invertase isolated from any two organisms (for example, yeast invertase vs. invertase from the small intestine of humans) show the same temperature optimum? Why or why not? Explain your answers below.

4 .		Explain why temperatures lower or higher than the optimum cause decreases in invertase activity. What is happening to the enzyme to produce these decreases in activity?

5 .		Carefully examine the curve for V_O vs. temperature. Is the slope of the line on both sides of the curve the same or different? If the slope of the line to the left of maximum velocity is different from the slope of the line to the right of maximum velocity, explain why this is. What is responsible for these differences in enzyme kinetics?

6 .		If you were to carry out these temperature experiments at a higher or lower [S], what effect would [S] have on the temperature optimum for invertase? Formulate a hypothesis and then test your hypothesis. What did you discover? Explain your results below.

7 .		If you were to carry out these temperature experiments at a higher or lower pH value, what effect would this have on the temperature optimum for invertase? Formulate a hypothesis and then test your hypothesis. What did you discover? Explain your results below.