Investigation into the Catalytic Role for the Tryptophan Residues within Domain III of Pseudomonas aeruginosa Exotoxin A

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• 作者：Bryan K. Beattie ; Gerry A. Prentice ; and A. Rod Merrill
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：December 3, 1996
• 年：1996
• 卷：35
• 期：48
• 页码：15134 - 15142
• 全文大小：389K
• 年卷期：v.35,no.48(December 3, 1996)
• ISSN：1520-4995

文摘

The role of the tryptophan residues in thesubstrate-binding and catalytic mechanism of anenzymatically active C-terminal fragment of Pseudomonasaeruginosa exotoxin A was studied byindividually or jointly replacing these residues with phenylalanine.Substitution of W-466 decreased theADP-ribosyltransferase and NAD⁺-glycohydrolase activitiesby 20- and 3-fold, respectively. In contrast,substitution of W-417 or W-558 with phenylalanine both resulted in a3-fold decrease in ADP-ribosyltransferase activity with, however, only a decrease by 40% and70% in NAD⁺-glycohydrolaseactivity, respectively. Simultaneous replacement of W-466 andW-558 resulted in a 200-fold decrease inADP-ribosyltransferase and an 6-fold decrease inNAD⁺-glycohydrolase activities, suggesting thatW-466may play a minor role in the transfer of ADP-ribose to the eEF-2protein. Chemical modification of thetryptophan residues in the wild-type toxin fragment byN-bromosuccinimide revealed the presence of asingle residue important for enzymatic activity, W-466, with a minorcontribution from W-558.Additionally, tryptophan residues, W-305 and W-417, wererefractory to oxidation by N-bromosuccinimide,which likely indicated the buried nature of these residues within theprotein structure. Titration of thewild-type toxin fragment with NAD⁺ resulted in thequenching of the intrinsic tryptophan fluorescenceto 58% of the initial value. Titration of the various single anda double tryptophan replacement mutantprotein(s) indicated that W-558 and W-466 are responsible for thesubstrate-induced fluorescence quenching,with the former being responsible for the largest fraction of theobserved quenching in the wild-typetoxin. Consequently, a molecular mechanism is proposed for thesubstrate-induced fluorescence quenchingof both W-466 and W-558. Furthermore, molecular modeling of therecent crystal structures for bothexotoxin A (domain III fragment) and diphtheria toxin, combined with avariety of previous results, hasled to the proposal for a catalytic mechanism for theADP-ribosyltransferase reaction. This mechanismfeatures a S_N1 attack (instead of the previously purportedS_N2 mechanism) by the diphthamide residue(nucleophile) of eukaryotic elongation factor 2 on the C-1 of thenicotinamide ribose of NAD⁺, whichresults in an inversion of configuration likely due to stericconstraints within theNAD⁺-toxin-elongationfactor 2 complex.