title: Generation of a catalytic antibody by site-directed mutagenesis.
abstract: A hybrid Fv fragment of the dinitrophenyl-binding immunoglobulin A (IgA), MPOC315, has been generated by reconstituting a recombinant variable light chain (VL) produced in Escherichia coli with a variable heavy chain (VH) derived from the antibody. The Tyr34 residue of VL 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 pH 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.
authors.affiliation: Department of Chemistry, University of California, Berkeley 94720.
authors.forename: E
authors.initials: E
authors.lastname: Baldwin
authors.forename: P G
authors.initials: PG
authors.lastname: Schultz
mesh_terms: D019297:2,4-Dinitrophenol; D000595:Amino Acid Sequence; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002384:Catalysis; D004140:Dinitrophenols / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D006868:Hydrolysis; D007070:Immunoglobulin A / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000138:chemical synthesis*
chemical_list: D004140:Dinitrophenols; D007070:Immunoglobulin A; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019297:2,4-Dinitrophenol