Conformational effects in biological catalysis: An antibody-catalyzed oxy-cope rearrangement

Antibody AZ-28 was generated against the chairlike transition-state analogu e (TSA) 1 and catalyzes the oxy-Cope rearrangement of substrate 2 to produc t 3. The germline precursor to AZ-28 catalyzes the reaction with a 35-fold higher rate (k(cat)/k(uncat) = 163000), despite a 40-fold lower binding aff inity for TSA.1 (K-D = 670 nM). To determine the structural basis for the d ifferences in the binding and catalytic properties of the germline and affi nity-matured antibodies, the X-ray crystal structures of the unliganded and TSA.1 complex of antibody AZ-28 have been determined at 2.8 and 2.6 Angstr om resolution, respectively; the structures of the unliganded and TSA.1 com plex of the germline precursor to AZ-28 were both determined at 2.0 Angstro m resolution. In the affinity-matured antibody hapten complex the TSA is fi xed in a catalytically unfavorable conformation by a combination of van der Waals and hydrogen-bonding interactions. The 2- and 5-phenyl substituents of TSA.1 are almost perpendicular to the cyclohexyl ring, leading to decrea sed orbital overlap and decreased stabilization of the putative transition state. The active site of the germline antibody appears to have an increase d degree of flexibility-CDRH3 moves 4.9 Angstrom outward from the active si te upon binding of TSA.1. We suggest that this conformational flexibility i n the germline antibody, which results in a lower binding affinity for TSA. 1, allows dynamic changes in the dihedral angle of the 2-phenyl substituent along the reaction coordinate. These conformational changes in turn lead t o enhanced orbital overlap and increased catalytic rate. These studies Sugg est that protein and substrate dynamics play a key role in this antibody-ca talyzed reaction.