Kinetic constants

Kinetic constants K_m and K_cat for hydrolysis of p-nitroanilide (pNA) derivatives of leucine, methionine, lysine and arginine by AP56 and AP28 were determined using the spectrophotometric assay. Substrate concentrations for Leu-pNA hydrolysis were in the range 0.6–4.8 mM and 0.5–4.0 mM for AP28 and AP56, respectively. Lys-pNA hydrolysis by AP28 and AP56 was studied at the concentration range of 0.15–2.4 mM. Hydrolysis of Arg- and Met-pNA by both AP28 and AP56 was studied at concentrations 0.3–4.8 mM. The enzyme concentration used for Leu- and Lys-pNA hydrolysis was 5.4 x 10⁻⁸ M (1.5 and 3 μg/ml for AP28 and AP56, respectively) while Arg- and Met-pNA hydrolysis was studied at an enzyme concentration of 1.3x10^-7 M (3.7 and 7.5 μg/ml, respectively). Molar concentration of AP56 was calculated using the theoretical molecular mass value of 55 kDa [30]. K_m and V_max values were derived from Lineweaver–Burk plots.