Insight into the catalytic mechanism of Pseudomonas aeruginosa exotoxin A. Studies of toxin interaction with eukaryotic elongation factor-2.
The molecular nature of the protein-protein interactions between the catalytic domain from Pseudomonas aeruginosa exotoxin A (PE24H) and its protein substrate, eukaryotic elongation factor-2 (eEF-2) were probed using a fluorescence resonance energy transfer method. Single cysteine mutant proteins of PE24H were prepared and site-specifically labeled with the donor fluorophore IAEDANS (5-(2-iodoacetylaminoethylamino)-1-napthalenesulfonic acid), whereas eEF-2 was labeled with the acceptor fluorophore fluorescein. The association was found to be independent of ionic strength and of the co-substrate, NAD(+) but dependent upon pH. The lack of requirement for NAD(+) to produce the toxin-eEF-2 complex demonstrates that the catalytic process is a random order mechanism, thereby disputing the current model. The previously observed pH dependence for catalytic function can be assigned to the toxin-eEF-2 binding event, as the pH dependence of binding observed in this study showed a strong correlation with enzymatic activity. The ability of the toxin to bind eEF-2 with bound GTP/GDP was assessed using nonhydrolyzable analogues. The results from the substrate binding and catalytic activity experiments indicate that PE24H is able to interact and bind with eEF-2 in all of its guanyl nucleotide-induced conformational states. Thus, the toxin ribosylates eEF-2 regardless of the nucleotide-charged state of eEF-2. These results represent the first detailed characterization of the molecular details and physiological conditions governing this protein-protein interaction.
Armstrong
Souzan
S
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario N1G 2W1, Canada.
Yates
Susan P
SP
Merrill
A Rod
AR
D036002:ADP Ribose Transferases / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D004735:Energy Transfer; D005098:Exotoxins / Q000378:metabolism*; D005453:Fluorescence; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000494:pharmacology; D020652:Peptide Elongation Factor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D037521:Virulence Factors / Q000378:metabolism*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D009243:NAD; D020652:Peptide Elongation Factor 2; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A