Investigation into the catalytic role for the tryptophan residues within domain III of Pseudomonas aeruginosa exotoxin A.
The role of the tryptophan residues in the substrate-binding and catalytic mechanism of an enzymatically active C-terminal fragment of Pseudomonas aeruginosa exotoxin A was studied by individually or jointly replacing these residues with phenylalanine. Substitution of W-466 decreased the ADP-ribosyltransferase and NAD(+)-glycohydrolase activities by 20- and 3-fold, respectively. In contrast, substitution of W-417 or W-558 with phenylalanine both resulted in a 3-fold decrease in ADP-ribosyltransferase activity with, however, only a decrease by 40% and 70% in NAD(+)-glycohydrolase activity, respectively. Simultaneous replacement of W-466 and W-558 resulted in a 200-fold decrease in ADP-ribosyltransferase and an 6-fold decrease in NAD(+)-glycohydrolase activities, suggesting that W-466 may play a minor role in the transfer of ADP-ribose to the eEF-2 protein. Chemical modification of the tryptophan residues in the wild-type toxin fragment by N-bromosuccinimide revealed the presence of a single residue important for enzymatic activity, W-466, with a minor contribution from W-558. Additionally, tryptophan residues, W-305 and W-417, were refractory to oxidation by N-bromosuccinimide, which likely indicated the buried nature of these residues within the protein structure. Titration of the wild-type toxin fragment with NAD+ resulted in the quenching of the intrinsic tryptophan fluorescence to 58% of the initial value. Titration of the various single and a double tryptophan replacement mutant protein(s) indicated that W-558 and W-466 are responsible for the substrate-induced fluorescence quenching, with the former being responsible for the largest fraction of the observed quenching in the wild-type toxin. Consequently, a molecular mechanism is proposed for the substrate-induced fluorescence quenching of both W-466 and W-558. Furthermore, molecular modeling of the recent crystal structures for both exotoxin A (domain III fragment) and diphtheria toxin, combined with a variety of previous results, has led to the proposal for a catalytic mechanism for the ADP-ribosyltransferase reaction. This mechanism features a SN1 attack (instead of the previously purported SN2 mechanism) by the diphthamide residue (nucleophile) of eukaryotic elongation factor 2 on the C-1 of the nicotinamide ribose of NAD+, which results in an inversion of configuration likely due to steric constraints within the NAD(+)-toxin-elongation factor 2 complex.
Beattie
B K
BK
Guelph-Waterloo Centre for Graduate Work in Chemistry, Ontario Department of Chemistry and Biochemistry, University of Guelph, Canada.
Prentice
G A
GA
Merrill
A R
AR
D036002:ADP Ribose Transferases / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D002384:Catalysis; D018360:Crystallography, X-Ray; D005098:Exotoxins / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism; D010084:Oxidation-Reduction; D010649:Phenylalanine; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D001974:Bromosuccinimide; D005098:Exotoxins; D009243:NAD; D009244:NAD+ Nucleosidase; D010649:Phenylalanine; D014364:Tryptophan; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A