PH INFLUENCES ON THE CRYSTAL-STRUCTURES AND MECHANISTIC PROPERTIES OFA HYDROLYTIC ANTIBODY

Antibody CNJ206 catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and product specificity. We report here k inetic and affinity measurements for this antibody in the pH 4-9.5 ran ge. We also report the X-ray structures of CNJ206 Fab liganded with a transition-state analog (TSA) and with one of the products of the hydr olysis, p-nitrophenol, at pH 4. This product binds in the same hydroph obic pocket as the p-nitrophenyl part of the TSA; its release at basic pH, where catalysis is usually measured, is facilitated by its ionize d state and this avoids product inhibition. The conformation of the CN J206 combining site is identical in this structure and in the previous ly determined structure of a CNJ206 Fab . TSA complex at pH 8. Taken t ogether with the pH-profile of the rate enhancement by CNJ206, the str uctural data support a mechanism for catalysis by CNJ206 involving sta bilization of a tetrahedral oxyanion intermediate by the peptide NH gr oups of two consecutive residues in the H3 complementarity-determining region.