DISSECTION OF AN ANTIBODY-CATALYZED REACTION

Antibody 43C9 accelerates the hydrolysis of a p-nitroanilide by a fact or of 2.5 x 10(5) over the background rate in addition to catalyzing t he hydrolysis of a series of aromatic esters. Since this represents on e of the largest rate accelerations achieved with an antibody, we have undertaken a series of studies aimed at uncovering the catalytic mech anism of 43C9. The immunogen, a phosphonamidate, was designed to mimic the geometric and electronic characteristics of the tetrahedral inter mediate that forms upon nucleophilic attack by hydroxide on the amide substrate. Further studies, however, revealed that the catalytic mecha nism is more complex and involves the fortuitous formation of a covale nt acyl-antibody intermediate as a consequence of complementary side c hain residues at the antibody-binding site. Several lines of evidence indicate that the catalytic mechanism involves two key residues: His-L 91, which acts as a nucleophile to form the acyl-antibody intermediate , and Arg-L96, which stabilizes the anionic tetrahedral moieties. Supp ort for this mechanism derives from the results of site-directed mutag enesis experiments and solvent deuterium isotope effects as well as di rect detection of the acyl-antibody by electrospray mass spectrometry. Despite its partial recapitulation of the course of action of enzymic counterparts, the reactivity of 43C9, like other antibodies, is appar ently limited by its affinity for the inducing immunogen. To go beyond this level, one must introduce additional catalytic functionality, pa rticularly general acid-base catalysis, through either improvements in transition-state analog design or site-specific mutagenesis.