Expression of enzymic activity by exotoxin A from Pseudomonas aeruginosa.
Exotoxin A from Pseudomonas aeruginosa is a single polypeptide chain (M(r), 66,000) containing little if any adenosine 5'-diphosphate ribosyltransferase or oxidized nicotinamide adenine dinucleotide glycohydrolase activity. These activities have been demonstrated in the reduced intact toxin and in a peptide (M(r), 26,000) isolated from culture fluids or toxin preparations after storage. In this report we describe methods for generating enzymically active fragments by cleaving the fully or partially reduced exotoxin by proteolytic or chemical methods. Incubation of reduced toxin with chymotrypsin in the presence of oxidized nicotinamide adenine dinucleotide yielded an enzymically active peptide (M(r), 26,000) similar to the fragment characterized previously. Chemical cleavage by treatment of the reduced molecule with CNBr or 2-nitro-5-thiocyanobenzoate yielded fragments (M(r), 50,000 and 30,000, respectively) with similar activities. Also both adenosine 5'-diphosphate ribosyltransferase and oxidized nicotinamide adenine dinucleotide glycohydrolase activities were maximally expressed by the intact exotoxin after reduction of only two of its four disulfide bridges. Kinetic constants for activated whole toxin were similar to those of fragment A of diphtheria toxin. It is evident that in the native toxin the catalytic center is buried or distorted and that alterations in the covalent structure permit the center to become exposed or assume an active configuration. It is unknown whether reduction, proteolytic processing, or both occur during the course of toxin action on whole cells.
D002250:Carbon Radioisotopes; D002918:Chymotrypsin / Q000494:pharmacology*; D004167:Diphtheria Toxin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005098:Exotoxins / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000378:metabolism; D009536:Niacinamide; D009713:Nucleotidyltransferases / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D002250:Carbon Radioisotopes; D004167:Diphtheria Toxin; D005098:Exotoxins; D009536:Niacinamide; D011065:Poly(ADP-ribose) Polymerases; D009713:Nucleotidyltransferases; D009244:NAD+ Nucleosidase; D002918:Chymotrypsin