Thermodynamic and Structural Basis for Transition-State Stabilization in Antibody-Catalyzed Hydrolysis

Catalytic antibodies 6D9 and 9C10, which were induced by immunization with a haptenic transition-state analog (TSA), catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. These antibodies stabilize the transition state to catalyze the hydrolysis reaction, strictly according to the theoretical relationship: for 6D9, kcat/kuncat = 895 and KS/KTSA = 900, and for 9C10, kcat/kuncat = 56 and KS/KTSA = 60. To elucidate the molecular basis of the antibody-catalyzed reaction, the crystal structure of 6D9 was determined, and the binding thermodynamics of 6D9 and 9C10 with both the substrate and the TSA were analyzed using isothermal titration calorimetry. The crystal structure of the unliganded 6D9 Fab was determined at 2.25 Å resolution and compared with that of the TSA-liganded 6D9 Fab reported previously, showing that the TSA is bound into the hydrophobic pocket of the antigen-combining site in an “induced fit” manner, especially at the L1 and H3 CDR loops. Thermodynamic analyses showed that 6D9 binds the substrate of the TSA with a positive ΔS, differing from general thermodynamic characteristics of antigen–antibody interactions. This positive ΔS could be due to the hydrophobic interactions between 6D9 and the substrate or the TSA mediated by Trp H100i. The difference in ΔG between substrate and TSA-binding to 6D9 was larger than that to 9C10, which is in good correlation with the larger kcat value of 6D9. Interestingly, the ΔΔG was mainly because of the ΔΔH. The correlation between kcat and ΔΔH is suggestive of “enthalpic strain” leading to destabilization of antibody–substrate complexes. Together with X-ray structural analyses, the thermodynamic analyses suggest that upon binding the substrate, the antibody alters the conformation of the ester moiety in the substrate from the planar Z form to a thermodynamically unstable twisted conformation, followed by conversion into the transition state. Enthalpic strain also contributes to the transition-state stabilization by destabilizing the ground state, and its degree is much larger for the more efficient catalytic antibody, 6D9.