Investigations of the ADP-ribosylation reaction catalyzed by Pseudomonas aeruginosa exotoxin A.
'Pseudomonas aeruginosa' exotoxin A (ETA) is member of the family of bacteria ADP-ribosylating toxins that use NAD+ as the ADP-ribose donor. The reaction catalyzed by ETA involves the nucleophilic attack of the diphthamide residue on the anomeric carbon of the nicotinamide ribose forming a new glycosidic bond. A fluorimetric assay involving the use of etheno-[beta]-nicotinamide adenine dinucleotide ([epsilon]-NAD+), an analogue of NAD+, has been developed which provides a procedure for assessing the kinetic parameters of this class of enzymes. Application of this new assay facilitated the determination of the kinetic parameters for the protein substrate of ETA, elongation factor 2 (EF-2), which have previously been difficult to measure. Ribosylated EF-2 blocks the growth of nascent polypeptide chain, hence, inhibiting protein synthesis leading to cell death. Antibiotic treatments of infection by 'Pseudomonas aeruginosa' are often ineffective in eradication; as a result, ETA has been a target for development of potent and selective inhibitors. The inhibition of enzymatic activity of ETA was evaluated by the above-mentioned assay. Here we demonstrate the effect of a series of compounds that inhibit the ADPRT activity of ETA. The type of inhibition by 1,8-naphthalimide, a potent inhibitor of ETA, was also investigated using this assay. A detailed knowledge of the molecular events in the catalytic activity of ETA requires a direct assay in which binding events of EF-2 to ETA can be monitored. In this study we investigated the use of the extrinsic fluorescent probe IAEDANS as a molecular reporter for the interaction between ETA and EF-2. Single Cys mutants of the catalytic domain of ETA, namely PE24, were modified with IAEDANS to introduce an extrinsic fluorescent probe in the protein structure at specific sites. EF-2, on the other hand, was chemically modified with fluorescein. The fluorescence energy transfer phenomenon between these two probes was used as a means of monitoring the binding interactions between the two proteins. The assay developed in the current investigation is the first direct method of monitoring the protein-protein interactions in this system.