Bifunctional catalysis of proton transfer at an antibody active site

The structure of antibody 13G5, which catalyzes the conversion of unactivated enzisoxazoles to the corresponding salicylonitriles, was determined at 2.65 angstrom resolution in the absence of ligand. It shows that the 2-aminobenzimidazolium derivative originally used for immunization was successful in templating an active site containing multiple functional groups. Thus, the side chains of three polar residues, Asp(H35), His(H95), and Glu(L34), project into an otherwise hydrophobic cavity. Site-directed mutagenesis and subsequent kinetic analysis identified Asp(H35) as the likely catalytic base that initiates proton abstraction. His(H95) appears to activate the base, whereas Glu(L34) may stabilize the incipient phenolate anion in the transition state at acidic pH. Introduction of alanine at position L34 unexpectedly boosts the activity of the catalyst substantially and broadens its pH optimum, perhaps because ordered water molecules are able to assume the role of proton donor over a wider range of conditions than the original glutamic acid. The aspartate base in the Glu(L34) Ala variant provides an astonishing 10(9)-fold rate advantage over the nonenzymatic reaction with acetate as base and exhibits an effective molarity of >10(6) M. These large effects illustrate the utility of bifunctional catalysis in an antibody active site for promoting reactions with unactivated substrates.