Generation of a Catalytic Antibody by Site-Directed Mutagenesis

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Abstract
A hybrid Fv fragment of the dinitrophenyl-binding immunoglobulin A (IgA), MOPC315, has been generated by reconstituting a recombinant variable light chain (V L ) produced in Escherichia coli with a variable heavy chain (V H ) derived from the antibody. The Tyr 34 residue of V L was substituted by His in order to introduce a catalytic imidazole into the combining site for the ester hydrolysis. The His mutant Fv accelerated the hydrolysis of the 7-hydroxycoumarin ester of 5-(2,4-dinitrophenyl)-aminopentanoic acid 90,000-fold compared to the reaction with 4-methyl imidazole at p H 6.8 and had an initial rate that was 45 times as great as that for the wild-type Fv. The hydrolyses of aminopropanoic and aminohexanoic homologs were not significantly accelerated. Thus a single deliberate amino acid change can introduce significant catalytic activity into an antibody-combining site, and chemical modification data can be used to locate potential sites for the introduction of catalytic residues.