A practical consideration for the substrate concentration when determining IC 50 values for enzyme inhibition

Determination of IC 50 values at a fixed substrate concentration ([S]) is frequently used to rank the potency of enzyme inhibitors and estimate inhibitor concentrations ([I]) to use in full inhibition analyses, particularly for structure-activity studies wherein the mode of inhibition is often known. Assays at an [S] yielding the greatest difference between the initial rates observed in the absence ( v o ) and in the presence ( v i ) of an inhibitor (i.e., v o – v i ) will increase the sensitivity for the detection of enzyme inhibition. For noncompetitive and uncompetitive inhibitors of single-substrate enzymes, v o – v i increases with increasing [S]; however, for competitive and linear mixed-type (LMT) inhibitors, v o – v i obtains a maximum at a specific “optimal” substrate concentration ([S] opt ). Equations are derived describing the dependence of [S] opt on [I], the dissociation constant for the inhibitor ( K i ), and the Michaelis constant for the substrate ( K m ). For example, for competitive inhibition, [S] opt = K m [Formula: see text]. For [I]/ K i values typically employed for inhibition studies (e.g., 0.5 ≤ [I]/ K i ≤ 4), [S] ≈ 2 K m or 3 K m will generally maximize the v o – v i difference for competitive or LMT (α ≥ 7) inhibitors, respectively. For competitive inhibition of bireactant enzymes, the “optimal” substrate concentrations depend on the Michaelis constants for both substrates, [I]/ K i , and the concentration of the second substrate.