I'm presently attempting to assay the activity of dihydroorotate dehydrogenase (DHODH) from two different species in the presence of a couple different inhibitors. For one DHODH, I already performed Michelis-Menton analyses and determined its Km for both dihydroorotate (substrate) and decylubiquinone (coenzyme, electron acceptor), and Vmax at 31 nM enzyme. I have also determined the IC50 of both inhibitors for this first DHODH at a single concentration of the substrate and coenzyme. For the other DHODH, I am limited to using only 10 nM of the enzyme in the assay, which is enough to see activity but prevents an apples-to-apples comparison. The ultimate goal is to compare the potency of inhibition of the same inhibitor between these two enzymes. As such, I'm wondering how to go about selecting the concentrations of dihydroorotate (DHO) and decylubiquinone (Qd) such that we have the closest we can get to an apples-to-apples comparison. My current thought is that I should again determine the Km for DHO and Qd with the second DHODH at the 10 nM concentration, but then keep the ratios of [DHO] and [Qd] to Km the same as they were in the experiment already performed for the first DHODH. However, I'm very curious to here what you all have to say.
Thank you in advance!
If the inhibitors are not tight binders (which means that the IC50s are much higher than the enzyme concentrations), the way to compare the potencies of a single inhibitor against 2 different enzymes is to measure the Kis of the inhibitors.
There are 2 ways of doing that using the enzyme reaction.
1. Full Michaelis-Menten enzyme kinetics analysis - Measure initial rates at a single enzyme concentration while varying the dihydroorotate (a), decylubiquinone (b) and inhibitor (c) concentrations in an a x b x c matrix of concentrations, then fitting the rate data to the kinetic rate equation for the inhibited reaction using nonlinear regression. This is a complicated experiment, but it is the best method. It also allows you to determine the mode of inhibition, if you don't already know it.
2. At fixed concentrations of enzyme, dihydroorotate and decylubiquinone, vary the inhibitor concentration to measure the IC50. Then use the relevant Cheng-Prusoff equation (derived from the kinetic rate equations with and without inhibitor) to calculate the Ki from the IC50. For this method, you need to have already measured the Kms for dihydroorotate, and decylubiquinone, and you need to know the kinetic rate equations with and without the inhibitor.
You may also be able to directly measure the dissociation constant of the inhibitor by any of several biophysical methods (e.g. isothermal titration calorimetry, intrinsic fluorescence, 2-D NMR). For this approach, you should know which form(s) of the enzyme the inhibitor binds to (the enzyme with no substrate bound, with one of the substrates bound, or with both bound).
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