ANALYSIS OF HAPTEN BINDING AND CATALYTIC DETERMINANTS IN A FAMILY OF CATALYTIC ANTIBODIES

We report here the cloning and kinetic analysis of a family of catalyt ic antibodies raised against a common transition state (TS) analog hap ten, which accelerate a unimolecular oxy-Cope rearrangement. Sequence analysis revealed close homologies among the heavy chains of the catal ytically active members of this set of antibodies, which derive mainly from a single germline gene, whereas the light chains can be traced b ack to several different, but related germline genes. The requirements for hapten binding and catalytic activity were determined by the cons truction of hybrid antibodies. Characterization of the latter antibodi es again indicates a strong conservation of binding site structure amo ng the catalytically active clones. The heavy chain was found to be th e determining factor for catalytic efficiency, while the light chain e xerted a smaller modulating effect that depended on light chain gene u sage and somatic mutations. Within the heavy chain, the catalytic acti vity of a clone, but not hapten binding affinity, depended on the sequ ence of the third complementarity determining region (CDR). No correla tion between high affinity for the hapten and high rate enhancement wa s found in the oxy-Cope system, a result that stands in contrast to th e expectations from transition state theory. A mechanistic explanation for this observation is provided based on the three-dimensional cryst al structure of the most active antibody, AZ-28, in complex with the h apten. This study demonstrates the utility of catalytic antibodies in examining the relationship between binding energy and catalysis in the evolution of biological catalysis, as well as expanding our understan ding of the molecular basis of an immune response. (C) 1998 Academic P ress Limited.