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Science 8 September 1989:
Vol. 245. no. 4922, pp. 1104 - 1107
DOI: 10.1126/science.2672338
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Articles

Science, Vol 245, Issue 4922, 1104-1107
Copyright © 1989 by American Association for the Advancement of Science

articles

Generation of a catalytic antibody by site-directed mutagenesis

E Baldwin and PG Schultz

Department of Chemistry, University of California, Berkeley 94720.

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.


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