Enzyme kinetics with Eisenthal & Cornish-Bowden (part II)
Back in October (2006) I wrote about this when I was messing around with enzyme kinetics after being asked by one of my professors to look into the Eisenthal & Cornish-Bowden method.
It’s basically a linearization of the Michaelis-Menten equation that lets one calculate some kinetic parameters right on the spot, literally.
And the best part of Eisenthal & Cornish-Bowden’s method is that there isn’t really any calculations to be done. It’s easy as drawing up the graph and you’re done!
OK, so if you’re not up to reading the original paper, then let me explain (in some simple lines) how you can use this great method to determine the kinetic parameters Vmax and Km.
- You need some experimental results for a constant concentration of enzyme [Eo], namely the concentrations of substrate [S] and the corresponding velocity v.
- Now with these results you draw up your plot. A horizontal line (X axis) for Km and a vertical line (Y axis) for Vmax.
- You then consider the negative value of your substrate concentrations to be your Km, so mark it on the horizontal line. And mark your corresponding velocity on the Vmax vertical axis. Draw a line through those two points.
- Do the same with every group of [S] & v. You end up with something like this:
If your experimental results were PERFECT, you’d get something like this. The intersection of all the lines would give you your Km and Vmax, directly!
Alright, we all know that experiments rarely go exactly as planned and the experimental results will most probably contain some error and not produce a single intersection. So a median value should be determined between the intersections found.
This method is also great for excluding outliers, because they usually don’t intersect with other lines.
This is a very empirical explanation of this method for ease of understanding. For more in depth information and how all this is done analytically, I’d recommend reading the original paper.
[Image from: James Zimmerman's Kinetics Background (.doc) ]
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