SITE-DIRECTED MUTAGENESIS OF A CATALYTIC ANTIBODY - AN ARGININE AND AHISTIDINE RESIDUE PLAY KEY ROLES

Individual residues important for ligand binding and catalytic activit y were identified by computer modeling and investigated by site-direct ed mutagenesis for catalytic antibody 43C9, which accelerates amide hy drolysis by a factor of 10(6). On the basis of a computer model, Tyr L 32, His L91, Arg L96, His H35, and Tyr H95 were chosen for replacement by site-directed mutagenesis. To facilitate these studies, an express ion system was developed in which properly folded 43C9 single-chain an tibody was secreted from an engineered Escherichia coil host. Substitu tion of His L91 by Gin produced a mutant with no catalytic activity, b ut whose affinities for ligands were nearly the same as those of the w ild-type, identifying His L91 as the nucleophile that forms the acyl i ntermediate implicated by previous kinetic studies. Arg L96 is also cr itical for catalytic activity and appears to function as an oxyanion h ole for the tetrahedral transition states. Two substitutions for His H 35 resulted in mutant proteins with no catalytic activity as well as a ltered affinities for ligands, indicating an important structural role for this residue. Substitutions for Tyr L32 and Tyr H95 were made in an attempt to improve the catalytic efficiency of 43C9. The results of these mutations allow us to propose a mechanism for 43C9-catalyzed hy drolysis: Substrate binding to 43C9 orients the scissile carbonyl grou p adjacent to both the His L91 and Arg L96 side chains. The imidazole of His L91 acts as a nucleophile, forming an acyl-antibody intermediat e that breaks down by hydroxide attack to afford the products and rege nerate the catalyst.