Abstract
Interestingly, 4A1, an antibody against p-nitrophenyl phosphonate, showed a significant rate acceleration against substrates that differ from the given haptenic structure in the carrier-proximal region. The rate acceleration (kcatkuncat) for one of the specific substrates is 6.4 × 104, 20-fold higher than that of a substrate congruent with the hapten. Kinetic analysis of Km and kcat values, as well as the affinity constant (Kd) values of the corresponding transition-state analogs, indicated that the rate enhancement is associated with a decrease in the activation energy due to stabilization of the transition -state in the cleavage reaction. In addition, the inactivation of 4A1 upon hydrolysis of a particular substrate was observed. The 600 MHz 13C NMR measurement clearly showed that the 13C-labeled fragment attached covalently to the 4A1 antibody, proving formation of an acyl-antibody. Further kinetic analysis study demonstrated that the 4A1 catalytic antibody uses a multistep kinetic sequence for the hydrolytic reaction.
