pH influences on the crystal structures and mechanistic properties of a hydrolytic antibody

Antibody CNJ206 catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and product specificity. We report here kinetic and affinity measurements for this antibody in the pH 4-9.5 range. 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 hydrolysis, p-nitrophenol, at pH 4. This product binds in the same hydrophobic pocket as the p-nitrophenyl part of the TSA; its release at basic pH, where catalysis is usually measured, is facilitated by its ionized state and this avoids product inhibition. The conformation of the CNJ206 combining site is identical in this structure and in the previously determined structure of a CNJ206 Fab•TSA complex at pH 8. Taken together with the pH-profile of the rate enhancement by CNJ206. the structural data support a mechanism for catalysis by CNJ206 involving stabilization of a tetrahedral oxyanion intermediate by the peptide NH groups of two consecutive residues in the H3 complementarity-determining region.