Since the main point of your study is to compare the activity of different mutants, the information you want is substrate Km and kcat. You will vary the substrate concentration and measure the initial rate of reaction in order to get Km and Vmax for each mutant and the wild-type using the Michaelis-Menten equation. From Vmax you will calculate kcat by dividing by the enzyme concentration.

It is crucial when performing this analysis that the rate of the primary enzyme reaction is rate-limiting, i.e. slower then the rate of the coupler. You can check this by showing that the initial rate is directly proportional to the enzyme concentration. If it isn't true, you need to use a higher coupling enzyme concentration.

You should also be careful to subtract any background rate at each substrate concentration by leaving out the primary enzyme. Use the purified enzyme if possible to minimize competing reactions.

If your enzyme catalyzes a reaction involving two or more substrates, things will get more complicated. You will have to vary both substrates, preferably in a matrix format.

Hi Kelly,

A great system to investigate!

In my MSc and the resultant paper years ago, I measured glutamate dehydrogenase (= also changes in NAD(P)/H abundance over 3min, and simply related the diff(Abs)/min to the amount of protein assayed. Since I investigated a native system, I blanked the reaction on the extraction buffer. https://www.researchgate.net/publication/226335472_Glutamate_dehydrogenase_of_the_germinating_triticale_seeds_gene_expression_activity_distribution_and_kinetic_characteristics

Also, I think you need to ensure your buffers do not cause the NADH to deteriorate much! And, needless to say, work out proper protein concentration / dilution activation, to work within the linear range of the activity.

I guess if the reaction's stoichiometry is kept for your coupled protein of interest, you could copy that algorithm, but in case anyone has done it differently, I'll gladly accrue a new method! ;) 

Article Glutamate dehydrogenase of the germinating triticale seeds: ...

Since the main point of your study is to compare the activity of different mutants, the information you want is substrate Km and kcat. You will vary the substrate concentration and measure the initial rate of reaction in order to get Km and Vmax for each mutant and the wild-type using the Michaelis-Menten equation. From Vmax you will calculate kcat by dividing by the enzyme concentration.

It is crucial when performing this analysis that the rate of the primary enzyme reaction is rate-limiting, i.e. slower then the rate of the coupler. You can check this by showing that the initial rate is directly proportional to the enzyme concentration. If it isn't true, you need to use a higher coupling enzyme concentration.

You should also be careful to subtract any background rate at each substrate concentration by leaving out the primary enzyme. Use the purified enzyme if possible to minimize competing reactions.

If your enzyme catalyzes a reaction involving two or more substrates, things will get more complicated. You will have to vary both substrates, preferably in a matrix format.

Kelly....

Follow the procedure ..

Reaction mixture: Buffer (of ur choice) + Enzyme source + NADH + other substrate (optional)

                           i.e., Buffer (0.85 ml) + Enzyme (0.1 ml depend on concentration of enzyme protein) + NADH (0.05 ml of 10 mM stock sigma)

Immediately put the above mixture in spectrophotometer and note the decrease in absorbance for every 30 sec for 3 min.

Calculate enzyme activity as follows:

Activity = Change in absorbance / (Extinction coefficient for NADH (in mM) * Time of incubation * mg of protein in enzyme source)

Remember: Enzyme should be fresh for measuring dehydrogenase activity...

You will find details about how to measure this activity in Bonete MJ and co-workers papers (They have been working on different dehydrogenases from haloarchaea for a long time). 

The enzyme and the NADH solution should be fresh for measuring the enzymatic activity and you have to calculate the extinction coefficient for NADH when you change the buffers used in the reaction mixtures.

I completely agree Adam B. Shapiro: It is crucial when performing this analysis that the rate of the primary enzyme reaction is rate-limiting, i.e. slower then the rate of the coupler. You can check this by showing that the initial rate is directly proportional to the enzyme concentration. If it isn't true, you need to use a higher coupling enzyme concentration.