DESIGN AND EXPRESSION OF ORGANOPHOSPHORUS ACID ANHYDRIDE HYDROLASE ACTIVITY IN HUMAN BUTYRYLCHOLINESTERASE

Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OF) nerve agents. To overcome this limitation, we selected several residues that were predicted to be within 3-10 Angst rom of both the active site Ser O-gamma and the oxyanion hole of human butyrylcholinesterase for mutation to His (G115H, G117H, Q119H, and G 121H). In remarkable contrast with wild-type (WT) and all other His mu tants tested, G117H underwent spontaneous reactivation following OP in hibition to regain 100% of original esterase activity with maximum ks values of approximately 6.8 x 10(-5) and 16 x 10(-5) s(-1) for GB (sar in) and VX, respectively, in 0.1 M Bis-Tris, 25 degrees C. The free en ergy of activation for k(3) was 19 kcal mol(-1), and measurement of pH dependence suggested that reactivation resulted from an acidic group with pK(a) 6.2. To evaluate further the importance of His in achieving this result, we changed the same Gly to Lys (G117K) and compared its substrate and inhibitor kinetics with those of G117H. Both mutants ret ained esterase activity with K-m values similar to those of WT for neu tral ester hydrolysis, but G117K did not reactivate. Complete reactiva tion proves that G117H is not irreversibly inhibited but instead funct ions as a catalyst for OP hydrolysis. Dephosphonylation is the rate-li miting step, and G117H effects overall rate constant enhancements of a pproximately 100- and 2000-fold above the uncatalyzed hydrolysis of GB and VX, respectively, at pH 6.0, 25.0 degrees C. We conclude that an appropriately positioned imidazolium ion in the oxyanion hole catalyze s dephosphonylation and, thereby, confers a novel organophosphonus aci d anhydride hydrolase activity upon butyrylcholinesterase.