Most inhibitors work extremely fast when establishing their binding equilibrium with the enzyme. However, tight-binding inhibitors establish their equilibrium on a much slower time-scale. Therefore, these types of inhibitors are called time-independent inhibitors because they show a change in initial velocity with time. There are four types of interactions that can slow down the kinetics of an inhibitor. The first is when the enzyme doesn’t have an inhibitor at all. The second interaction is when the equilibrium constants are very small compared to the enzyme turnover when the inhibitor is reversible. The third interaction is when the inhibitor binds, it forms an EI complex that is then isomerized to form a new complex. This new complex significantly inhibits the reaction, therefore slowing it down. The fourth interaction deals with irreversible inhibitors that can act as affinity labels for the enzyme, therefore slowing the reaction down based on its mechanism. Two examples of time-dependent inhibitors are serine proteases and the prostaglandin G/H synthase.
Determining reversibility involves performing a large dilution, dialysis, filter binding, or size exclusion chromatography. In order to tell the difference between covalent inactivation from noncovalent inhibition, it is important to be able to tell when the inhibitor is being released upon denaturation of the enzyme. For example, if there is an inhibitor that is covalent and it is denatured, then the inhibitor would still be attached to the denatured protein due to the covalent bonds between the inhibitor and the enzyme. However, if the inhibitor was noncovalent, then the active site of the enzyme would release the inhibitor in to the denaturing solution.
Copeland, Robert A. Enzymes. Wiley-VCH, Inc., 2000. 318-34.