Please mention the procedure. I have observed a time dependent inhibition in my experiment. References are bit confusing. I have read Robert Copeland, need a simple procedure, easy to comprehend.
You begin by collecting a set of progress curves for the enzyme reaction at various inhibitor concentrations. These reactions should be initiated by adding the enzyme; there should be no preincubation of the inhibitor with the enzyme. It is very helpful if you have a continuous readout of enzyme activity so that you can collect the entire progress curve with one sample.
The usual next step is to obtain a rate constant for inhibition (kobs) for each progress curve by fitting the progress curve to a particular equation (Eqn 9 in the attached paper by Peter Tonge) using nonlinear regression. Then, make a plot of kobs versus inhibitor concentration. This plot may be linear or hyperbolic. If it is linear, the slope is kinact/Ki,app. Obtaining individual values of kinact and Ki may be difficult in that case because the rate of inactivation is high while the inhibitor affinity is low. Another possible reason for a linear plot is that the inhibition follows a one-step mechanism instead of a two-step mechanism.
If the plot is curved, fit the data to the appropriate equation (Eqn 10) to obtain the individuals values of kinact and Ki,app.
For a competitive irreversible inhibitor, Ki,app = Ki(1+[S]/Km), so you need to also measure the substrate's Km.
There is another way to get kinact/Ki, which is to use numerical integration to perform a global fit of the whole set of progress curves at once. This can be done using a suitable computer program, such as Kintek Global Kinetic Explorer. This takes some effort to learn, but it is worth the time if you are expecting to do a lot of these measurements because it allows you to do the analysis quite quickly.
Amit Kumar Mukherjee: In tight binding inhibitors the binding is frequently covalent, but it can also result from non-covalent binding involving a slow conformational change in the enzyme. For example, in Na/K-ATPase the MgPi-complex analogue [CoIII(NH3)4PO4] binds tightly, but non-covalently (doi: 10.1111/j.1432-1033.1991.tb15720.x, 10.1111/j.1432-1033.1990.tb19346.x and 10.1016/S0022-5193(05)80546-6). Binding is then reversible by caotropic agents.
doi:10.1016/S0076-6879(77)46005-1 and 10.1016/S0076-6879(82)87027-4 give still useful overviews.
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