Esterase activity of catalytic IgG and IgM antibodies for the hydrolysis of p-nitrophenyl acetate

Abstract The esterase activities of IgG and IgM monoclonal antibodies (Antibody KD2-1 to KD2-260) prepared with a hapten of p -nitrophenyl hydrogen 4-(hydroxycarbonyl) butylphosphonate were examined in the hydrolysis of p -nitrophenyl acetate in 2.4–4.0% (v/v) MeCNH 2 O in TrisHCl buffer (pH 5.0–8.0) at 293–308 K. The most efficient Antibody KD2-260, which resulted in k cat = 2.53 (293 K)−7.91 min −1 (308 K), k cat / k uncat = 2.09 × 10 3 (308 K)−3.12 × 10 3 (293 K), and K m = 4.9 × 10 −6 (293 K)−1.5 × 10 −5 mol·dm −3 (308 K) at pH 6.0, was found to be catalytically more active than the Schultz antibody (Antibody 48G-7-4A1) prepared with the same hapten. The K m / K i ratio (80) obtained for the hydrolysis activity of Antibody KD2-260 at 303 K (pH 6.0) by the inhibition experiment, the value of which is corresponding to ( k cat / k uncat )/29, demonstrated the efficient Antibody KD2-260 catalyzed hydrolysis via the stabilization of the transition state (TS) of the reaction. The analogy between TS and TS analogue (or the hapten) in their electronic structures and the activation parameters was also discussed in connection with the esterase activity of Antibody KD2-260.