Steady-state kinetic analysis of reversible enzyme inhibitors: A case study on calf intestine alkaline phosphatase

Enzymes are important drug targets and inhibition of enzymatic activity is an important therapeutic strategy. Enzyme assays measuring catalytic activity are utilized in both the discovery and development of new drugs. Colorimetric assays based on the release of 4-nitrophenol from substrates are commonly used. 4-Nitrophenol is only partly ionized to 4-nitrophenolate under typical assay conditions (pH 7-9) leading to under-estimation of product formation rates due to the much lower extinction coefficient of 4-nitrophenol compared to 4-nitrophenolate. Determination of 4-nitrophenol pK_a values based on absorbance at 405 nm as a function of experimental pH values is reported, allowing for calculation of a corrected extinction coefficient at the assay pH. Characterization of inhibitor properties using steady-state enzyme kinetics is demonstrated using calf intestine alkaline phosphatase and 4-nitrophenyl phosphate as substrate at pH ∼8.2. The following kinetic parameters were determined: K_m= 40±3 µM; V_max= 72.8±1.2 µmolmin^-1mg protein^-1; k_cat= 9.70±0.16 s^-1; k_cat/K_m= 2.44±0.16 × 10⁵ M^-1s^-1 (mean± SEM, N = 4). Sodium orthovanadate and EDTA were used as model inhibitors and the following pIC₅₀ values were measured using dose-response curves: 6.61±0.08 and 3.07±0.03 (mean±SEM, N = 4). Rapid dilution experiments determined that inhibition was reversible for sodium orthovanadate and irreversible for EDTA. A K_i value for orthovanadate of 51±8 nM (mean±SEM, N = 3) was determined. Finally, data analysis and statistical design of experiments are discussed.