CELL BIOLOGY 220 JMU

CELL AND MOLECULAR BIOLOGY - BIO 214

ASSIGNMENT OF THE WEEK: #3

Enzyme Kinetics Tutorial

This exercise will allow you to use an interactive model in Excel to visualize the effects of changing the Michaelis Menten constant (Km) and maximum velocity (Vmax) on the rate of enzyme activity over a range of substrate concentrations. For more background information on these concepts, please consult your lecture textbook, the lab manual, or the following websites:

Enzyme Biochemistry Chapter – The MIT Biology Hypertextbook designed by the Experimental Study Group at Massachusetts Institute of Technology. This is a brief, thorough, and excellent overview of the topic. (This site is best viewed with Netscape or Firefox. It does not work with Internet Explorer

Enzyme Kinetics - from Kimball's Biology Pages.

Enzyme Kinetics Animations - created by undergraduates at Welseley College.

Introduction to enzyme kinetics - From curvefit.com.

Energy, Enzymes, and Catalysis Problem Set – developed by The Biology Project at the University of Arizona.

Answer form

For this assignment there is no printable answer form. Please type your answers in a separate Word file, print it and turn it in by the due date. You may work in pairs turning in one form with both names. Please identify the course, section, semester, and assignment number at the top of the page.

Definitions

Km is the Michaelis Menten constant. The Km of an enzyme for a specific substrate is the concentration of that substrate at which the rate of the reaction is half of the maximum rate.

Vmax is the theoretical maximum rate of the reaction. At very high [S] Vmax may be approached, but it can never be attained.

[S] is the concentration of substrate.

The model

Download the Excel file called kinetics_model.xls and open it.

Notice that near the top of the page is the Michaelis Menten equation in which the rate of reaction is a function of Km, Vmax and substrate concentration [S]. This equation is used below to calculate rates.

In the light blue box are values for two sets of Km and Vmax; one black set and one red set. These are the numbers you will change to answer the questions. Below the blue box are two blocks of data. The first block contains [S] data on the left and rates on the right. Notice that for each [S], two rates are given, one black and one red corresponding to the black and red kinetic constants in the blue box. The second block of data contains the inverse of all numbers in the first block and is used to generate a Lineweaver-Burk Plot. From this plot Km and Vmax can be calculated based on where the lines intercept each axis.

To the right of each block are figures illustrating the data in each block. The top figure contains plots of the reaction rates (black and red) vs. [S]. From this figure Km and Vmax can be estimated. The bottom figure contains the Lineweaver-Burk plot from which Km and Vmax can be calculated. Note that when the inverse is plotted, big numbers become small and small numbers become big!

Questions:

Assume the black and red sets of numbers represent the same type of enzyme from two different organisms. To see the effect of Km on reaction rate, set both Vmax values to 10, set the black Km value to 20 and the red Km value to 5.

1A. What effect does a high Km have on the rate of activity at low substrate concentration?

1B. What effect does a high Km have on the rate of activity at high substrate concentration?

1C. On which axis of the Lineweaver-Burk plot do the two lines intersect (cross)? It is at this point that Vmax can be calculated.

Now visualize the effect of Vmax on reaction rate. Set the Km values for both enzymes to 5, and set the black Vmax to 6 and the red Vmax to 10.

2A. What effect does a high Vmax have on the rate of activity at low substrate concentration?

2B. What effect does a high Vmax have on the rate of activity at high substrate concentration?

2C. On which axis of the Lineweaver-Burk plot do these two lines intersect (cross)? It is at this point that Km can be calculated.

3A. Find two sets of kinetic parameters in which at low [S] the black enzyme is more active and at high [S] the red enzyme is more active.
Black Vmax: ____ Red Vmax ____
Black Km: ____ Red Km ____

3B. Which parameter, low Km or high Vmax, leads to a higher rate of activity at low [S]?

3C. Which parameter, low Km or high Vmax, leads to a higher rate of reaction at high [S]?

For more fun with this model, find out what effect competitive and noncompetitive inhibitors have on enzymes and model them to see how cells might overcome their effects.

If you are not a JMU student and you use this model, please send feedback to Jon Monroe. Thanks!