ACTIVE-SITE MUTATIONS OF PSEUDOMONAS-AERUGINOSA EXOTOXIN-A - ANALYSISOF THE HIS(440) RESIDUE

Pseudomonas aeruginosa exotoxin A (ETA) is a member of the family of b acterial ADP-ribosylating toxins which use NAD(+) as the ADP-ribose do nor, By analogy to diphtheria and pertussis toxins, the His(440) resid ue of ETA has been proposed to be one of the critical residues within the active site of the toxin. In this study the role of the His(440) r esidue was explored through site-directed mutagenesis which resulted i n the production of ETA proteins containing Ala, Asn, and Phe substitu tions at the 440 position, The His(440) substituted ETA proteins were purified and analyzed. All substitutions at the 440 site displayed sev erely reduced ADP-ribosylation activity (>1000-fold). However, NAD gly cohydrolase activity remained intact and in the case of ETAH440N actua lly increased 10-fold. NAD(+) binding is not affected by substitutions at the 440 site as indicated by similar K-m values for the ETA varian ts tested. Conformational integrity of the mutant toxins appears to be largely unaffected as assessed by analysis with a conformation-sensit ive monoclonal antibody as well as sensitivity to proteinase digestion . In view of the location of His(440) residue within or close to the p roposed NAD(+)-binding site, these results suggest that His(440) may b e a catalytic residue involved in the transfer of the ADP-ribose moiet y to the EF-2 substrate.