Expression of FAS-independent ADP-ribosyltransferase activity by a catalytic deletion peptide of Pseudomonas aeruginosa exoenzyme S

Earlier studies reported that Pseudomonas aeruginosa exoenzyme S (ExoS) pos sessed an absolute requirement for the eukaryotic protein factor activating exoenzyme S (FAS) for expressing ADP-ribosyltransferase activity. During t he characterization of a serum-derived FAS-like activity, we observed the a bility of a catalytic deletion peptide of ExoS (Delta N222) to ADP-ribosyla te target proteins in the absence of FAS. Characterization of the activatio n of Delta N222 by FAS provided an opportunity to gain insight into the mec hanism of ExoS activation by FAS. Under standard enzyme assay conditions, t he initial rate of FAS-independent ADP-ribosyltransferase activity of Delta N222 was not linear with time and rapidly approached zero. Dilution into h igh-ionic strength buffers stabilized Delta N222 so it could express FAS-in dependent ADP-ribosyltransferase activity at a linear rate. This stabilizat ion was a general salt effect, since dilution into a 1.0 M solution of eith er NaCH3COOH, NaCl, or KCl stabilized the ADP-ribosyltransferase activity o f Delta N222. Kinetic analysis in a high-ionic strength buffer showed that FAS enhanced the catalytic activity of Delta N222 by increasing the affinit y for NAD and stimulating the turnover rate. Velocity experiments indicated that the stabilization of Delta N222 by high salt was not functionally ide ntical to stabilization by FAS. Together, these data implicate a dual role for FAS in the allosteric activation of ExoS, involving both substrate bind ing and catalysis.