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 ofp-trifluoroacetamidophenylacetic acid, demonstrating a structural correspondence with the phosphonate hapten.Saturation kinetics are observed and kinetic parameters (km., V.., and K.) are reported. The haptenic phosphonate is a competitive inhibitor of the reaction (K;, 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.The immune system is the most prolific source of receptor molecules known; yet, it remains largely unexploited for the study of the relationship of ligand binding to enzymic function. Antibodies offer the ability to specify binding interactions toward any molecule of theoretical interest, and the consequences of that interaction may then be investigated. The central tenet of biological catalysis ascribes enzymic rate accelerations to the changes in binding interactions along a reaction coordinate such that energy of binding, and thus stabilization ofthe complex, increases as bound substrates or products approach the bound transition state (1, 2). Evidence for this theory comes from the observation that substances that are thought to model the presumed transition states are often strongly bound to the enzymes as competitive inhibitors (3,4). Given the availability oftransition state analogs and the diversity of immunological receptors, one can ask whether it is possible to derive a chemical function from a pure binding function by selecting an antibody-antigen pair to define the optimal binding in a transition-state receptor complex.Immunological binding has been recognized as a potential basis for experimentally diverting binding interactions to catalytic processes (5). Sporadic attempts to introduce reactive groups into an antibody's combining site have been unsuccessful (6). Some monoclonal antibodies are reported to be fortuitously endowed with nucleophilic residues that allow a reaction with an activated ester appendage on a homologous hapten recognized by the antib...

Phosphonate Ester Probes for Proteolytic Antibodies

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