We report NMR and kinetic studies of antibody AZ-28, which was generated against the diaryl-substituted cyclohexanol derivative 3 and catalyzes the oxy-Cope rearrangement of the corresponding hexadiene 1 to aldehyde 2 (Braisted, A. C., Schultz, P. G. J. Ant. Chem. Sec. 1994, 116, 2211-2212). Conformational studies of free substrate and the antibody-substrate complex using transferred-NOE experiments demonstrate that the antibody binds the substrate in a cyclic conformation, consistent with the chair-like geometry of 3. In contrast, free substrate adopts an extended conformation in Solution. In spite of the conformational restriction of the bound substrate revealed by NMR, the temperature dependence of the reaction indicates that the antibody functions primarily by lowering Delta H double dagger, offsetting a decrease in Delta S double dagger; the values of Delta H double dagger and Delta S double dagger are 15.4 +/- 2.4 kcal/mol and -23 +/- 8 cal/mol K, respectively. A secondary kinetic isotope effect (k(cat)H/k(cat)D) of 0.61 +/- 0.1 was measured for substrate 23 in which both allylic termini are dideuterated, indicating that the chemical rearrangement step is wholly or partially rate limiting under saturating conditions. The magnitude of this secondary isotope effect is consistent with a significant degree of bond formation between C1 and C6 of the substrate in the transition state. These results, together with the recently reported three-dimensional crystal structure for the antibody-hapten 3 complex (Ulrich, H.; Mundroff, E.; Santarsiero, B. D.; Driggers, E. M.; Stevens, R. C.; Schultz, P. G. Nature 1997, 389; 271-275), provide a detailed mechanistic model for this antibody-catalyzed reaction.