A practical consideration for the substrate concentration when determining IC₅₀ values for enzyme inhibition

Determination of IC₅₀ 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 $\sqrt{1+\left(\left[I\right]/K_i\right)}$ . For [I]/K_i values typically employed for inhibition studies (e.g., 0.5 ≤ [I]/K_i ≤ 4), [S] ≈ 2K_m or 3K_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.