Kinetic and Mechanistic Investigation of a Hydrolytic Catalytic Antibody Having Remarkable Substrate Specificity: Substituent Effects on Catalysis.
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Monoclonal antibodies were raised against p-nitrophenyl phosphonate to elicit catalytic antibodies capable of hydrolyzing p-nitrophenyl carbonates. Of 34 clones selected, three clones catalyzed the hydrolysis of methyl p-nitrophenyl carbonate. Interestingly, 4A1, an antibody out of those clones, showed a significant rate acceleration against substrates that differ from the given haptenic structure in the carrier-proximal region. The rate acceleration (kcat/kuncat) 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 catalytic antibody 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.
- 公益社団法人 日本化学会の論文
公益社団法人 日本化学会 | 論文
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