My enzyme of interest does not require a co-factor such as NADPH/NADH. However, when used in a certain application, I discovered that NADP(H) (and possibly NAD(H)) inhibits my enzyme. I want to figure out the Ki of NADP(H) and what type of inhibition occurs (competitive, non-competitive, irreversible etc). Not quite sure where to get started :(
Additionally, I use a coupled enzyme assay which requires NADH to assay my protein... Any ideas on how to prevent interefence?
First, you will have to find a different way to assay your enzyme activity if NAD(H) acts as an inhibitor. If you are using a coupled assay, make absolutely sure that the coupling reaction is not rate limiting at any substrate and enzyme concentration you are using. Otherwise the rate measurements will be incorrect.
The textbook method for studying inhibitor kinetics is for inhibitors of enzymes with a single substrate. If your enzyme's reaction involves more than one substrate, the analysis is more complicated. Advanced enzymology textbooks can be consulted for the details. Two such books are linked below:
https://www.springer.com/gp/book/9780306467127
https://www.amazon.com/Enzyme-Kinetics-Behavior-Equilibrium-Steady-State/dp/0471303097
For an enzyme with a single substrate and a single inhibitor, the experimental method involves measuring the initial rate (V) of the reaction as you vary the substrate ([S]) concentration at several fixed concentrations of the inhibitor ([I]).
The old-fashioned way to analyze the data is then to make double-reciprocal plots (1/V versus 1/[S]) at each inhibitor concentration and to perform linear regressions. The appearance of these plots is diagnostic of the mode of inhibition (see any introductory biochemistry textbook). A replot of the data called a Dixon plot can then be used to measure the Ki.
A more modern way to analyze the data is to use a nonlinear regression computer program. The initial rate data are fit to kinetic rate equations describing the various possible modes of inhibition. The equation that fits the data best is the one that properly describes the mode. The values of all the kinetic constants (Vmax, Km, and Ki) are obtained from the fitting. These equations are to be found in many textbooks or on-line sources.
Just to add to Adam's comments you may also wish to look at the "direct plot" (doi:10.1042/bj1390721 and doi:10.1042/bj1390715). As this method is non-parametric it is simple, quick and also robust against measurement errors, which beginners are prone to make. In doi:10.1007/978-3-319-19920-7_6 I provide a worked-out example for this method.
Also, make sure the inhibition you are seeing is not due to inhibition of the coupling reaction by NADPH.
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