| Περίληψη: |
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 (vo) and in the presence (vi) of an inhibitor (i.e., vo – vi) will increase the sensitivity for the detection of enzyme inhibition. For noncompetitive and uncompetitive inhibitors of single-substrate enzymes, vo – vi increases with increasing [S]; however, for competitive and linear mixed-type (LMT) inhibitors, vo – vi 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 (Ki), and the Michaelis constant for the substrate (Km). For example, for competitive inhibition, [S]opt = Km 1 + [ I ] / K i . For [I]/Ki values typically employed for inhibition studies (e.g., 0.5 ≤ [I]/Ki ≤ 4), [S] ≈ 2Km or 3Km will generally maximize the vo – vi 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]/Ki, and the concentration of the second substrate. [ABSTRACT FROM AUTHOR] |