Chemical reactivity at an antibody binding site elicited by mechanistic design of a synthetic antigen.

Abstract

Monoaryl phosphonate esters, designated as analogs of the transition state in the hydrolysis of carboxylic esters, were synthesized and used as haptens to generate specific monoclonal antibodies. Some of these antibodies react with cognate aryl carboxylic esters to release a fluorescent alcohol. The reaction appears to be stoichiometric; however, the activity is slowly regenerated under alkaline conditions or by treatment with hydroxylamine. Specificity is rigorous for esters of p-trifluoroacetamidophenylacetic acid, demonstrating a structural correspondence with the phosphonate hapten. Saturation kinetics are observed and kinetic parameters (kmax, Vmax, and Km) are reported. The haptenic phosphonate is a competitive inhibitor of the reaction (Ki, 35 nM); whereas the carboxylate product of ester hydrolysis is a less effective inhibitor (Ki, ca. 7500 nM). Chemical modification of side chain groups in the protein show a partial reduction in activity on acylation of lysine or nitration of tyrosine and a dramatic quenching upon modification of histidine. The evidence is discussed in terms of a mechanism in which amino acids of the antibody combining site participate in nucleophilic and/or general base catalysis. The properties of this system suggest that it is an example of enzymic transacylation where a deacylation step is not catalyzed. The possibility of deriving enzymic function from immunological specificity through this approach is advanced.