[KernieBosar]

I'm taking biochemistry now and we are currently learning about enzymes, specifically their kinetics. I'm familiar with the idea of Vmax, Km, Kcat, Kcat/Km, Michaelis-Menton equation, and sort of the idea of a lineweaver burk plot. However, I can't figure out how to actually do one though, especially by hand.

My homework assignment goes like this:

We are given data obtained from an enzymatic reaction in the absence of, and also in the presence of 3 different inhibitors.

The data includes 10 different {S}, ranging from .2 to 5.00 (mM)

Initial velocities (1 without an 3 with inhibitor) in nM/min.
The velocities range from 5.56 to 83.33 and some are just really clustered.

We are to determine the Vmax and Km of the enzyme in the presence and absence of each inhibitor, calculate the Ki for each inhibitor, tell whether they are all the same inhibitor, figure out which is the best and why, determine catalytic efficiency and the turnover knowing that its concentration was 2 nM throughout the experiment, and lastly, what happens to efficiency and turnover with each inhibitor present.

I can't find any helpful examples of making a lineweaver burk plot; all of them are very simple, generic, and have a low range of numbers and most are done by computer. So far, videos from Moof university ahve helped, but not nearly enough.

Can anyone offer some advice on how to get started? I'm also really not sure how to set up the graph with such a wide range of values, especially if I want it to look good.

Thanks!

*MOD Edit: change brackets to braces to avoid HTML code*

[KernieBosar]

Additional question. Why does the bottom half of my post have a strike through through most of it? New to the forum!

[salteen]

Step 1 is to actually plot the data.  What is a Lineweaver-Burk plot?   I.e. what do each of your axes represent?

[KernieBosar]

http://en.wikipedia.org/wiki/Lineweaver%E2%80%93Burk_plot

So if you plot the data as is you will end up with a hyperbole. So, you instead take the reciprocal of the data (1/Vo, 1/{S}) and it gives you a straight line. The data for 1/Vo goes on the Y-axis while 1/{S]\} goes on the x axis. The point at which the data crosses the y-axis is 1/Vmax, the point at which the lien crosses the x -axis is - 1/Km.

I guess my main problem is deciding how to plot the data since there is such a wide range. For instance, data that goes on the y-axis  goes for Vo without an inhibitor is 0.012, 0.0125, 0.013, 0.014, 0.015, 0.02, 0.03, 0.04, 0.05, and 0.06. The Vo data for Inhibitor B is 0.036, 0.037, 0.039, 0.042, 0.045, 0.06, 0.09, 0.12, 0.15, 0.180

I am pretty bad at making graphs. How should I mark the axis for Y that will work well?

[salteen]

Are you allowed to use a computer?  If so, I usually use excel for these kinds of things.  Not only will it automatically scale the graph to the dimensions you need, you can also use linear regression to plot a line through your points.

[KernieBosar]

We cannot use a computer, everything has to be done by hand and we have to show all of our work!

I guess my main concern is having a graph that I can actually work with and isn't just clustered. For example, for the data involving Inhibitor B, the values are 0.036, 0.037, 0.039, 0.042, 0.045, 0.06, 0.09, 0.12, 0.15, 0.180. Will those lower values be too clustered to actually work with? I guess I can plot it increments of 0.006, but how can I notice a significant difference between 0.037, 0.039, 0.042, and 0.045... ?

[Arkcon]

I would us a computer to quickly see how the numbers look, and then plot it on graph paper by hand.  That's what I used to do in college.  Try to see if you can find decimal ruled graph paper -- there are dark line grids, with ten fine lines between each.  Use those thin lines for decimal fractions.  Using this sort of paper gives you much better control over the plot.  Looking at the numbers on a page and seeing the shape is difficult for most people.