Restoration of enzymic activity and cytotoxicity of mutant, E553C, Pseudomonas aeruginosa exotoxin A by reaction with iodoacetic acid.
Pseudomonas aeruginosa exotoxin A (ETA) is inactivated greater than 1,000-fold when an active site glutamic acid, E553, is mutated to aspartic acid (Douglas, C.M., and Collier, R. J. (1987) J. Bacteriol. 169, 4967-4971). To test the effect of creating a carboxyl-containing side chain at position 553 longer than that of glutamic acid, we first replaced Glu-553 with cysteine by site-directed mutagenesis of cloned ETA and then carboxymethylated the cysteine side chain with iodoacetic acid. The E553C mutation reduced ADP-ribosyltransferase and cytotoxic activities greater than 10,000-fold. Reaction of the mutant with iodoacetic acid enhanced enzymic activity 2,500-fold, to a level approximately one-sixth that of wild type toxin, and restored cytotoxicity to a slightly lesser extent. Iodoacetamide did not activate the mutant, and neither iodoacetic acid nor iodoacetamide affected the activity of wild type toxin. These results show that the carboxyl group of Glu-553 is important for ADP-ribosylation activity and imply flexibility in the enzyme-substrate complex in accommodating the slightly longer S-carboxymethylcysteine side chain. This general approach may have applications in protein engineering as well as in studying carboxyl side chain functions in enzymes.
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D001427:Bacterial Toxins; D002470:Cell Survival; D004926:Escherichia coli; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism*; D007163:Immunosorbent Techniques; D007461:Iodoacetates / Q000378:metabolism*; D019807:Iodoacetic Acid; D007700:Kinetics; D009154:Mutation; D010430:Pentosyltransferases / Q000378:metabolism*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose; D001427:Bacterial Toxins; D005098:Exotoxins; D007461:Iodoacetates; D019807:Iodoacetic Acid; D010430:Pentosyltransferases; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A