How to plan a classical enzyme-substrate reaction ???

10 October 2019 1 5K Report

Hello everyone!

I am mostly a structural biologist who did not get a chance yet to delve into a classical biochemical enzyme-substrate reaction.

So as things would have it, I will have to perform one now.

I have an NAD dehydrogenase enzyme and I have a substrate for it. As I understand, I have to monitor the decrease in the absorbance at 340 nm as NADH will be converted to NAD+

I plan to use the 96 microwell plate reader for this purpose.

How will I go about measuring the Vmax, Km, Kcat and the rate of reaction?

Also, how will I utilize the absorbance to obtain the rate of reaction? How about the enzyme and the substrate concentration?

I will be more than happy to get some help on this!

Thanks,

Nishit

Danial Yousefi

I suggest you to consult the literature regarding how to assay your particular enzyme, and details such as how to start and stop the reaction it catalyzes, as well as what to use as your negative control.

But in general, you need to measure the absorbance for samples of the enzymatic reaction that started with several different concentrations of the substrate and stopped at several different time intervals. The relationship between absorbance and concentration can be obtained by measuring the absorbance for a series of standard solutions of the substance you are going to measure; in your case, NADH. By the way, you may measure the peak of absorbance for your NADH sample experimentally using the particular spectrometer you are going to use. You need to know [E]0 as well, the concentration of the active enzyme at the beginning of the reaction.

Assuming that your reaction follows the Michaelis-Menten kinetics, the kinetic parameters can be easily calculated by data analysis software such as GraphPad Prism and SigmaPlot, which provide user-friendly wizards to obtain these parameters.

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