title: Toward the elucidation of the catalytic mechanism of the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A.
abstract: The catalytic mechanism for the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A was investigated by steady-state and stopped-flow kinetic analyses. The rate constants for binding of the NAD(+) substrate to the enzyme were found to be 4.7 +/- 0.4 microM(-1) s(-1) and 194 +/- 15 s(-1) for k(on) and k(off), respectively. The k(on) and k(off) rate constants for the eEF-2 substrate binding to the enzyme were 320 +/- 39 microM(-1) s(-1) and 131 +/- 22 s(-1), respectively. A potent, competitive inhibitor against the enzyme, 1,8-naphthalimide, bound the enzyme with k(on) and k(off) rates of 82 +/- 9 microM(-1) s(-1) and 51 +/- 6 s(-1), respectively. Furthermore, the binding on and off rates for the reaction products, ADP-ribose and nicotinamide, were too rapid for detection with the stopped-flow technique. Investigation of the pre-steady-state kinetics for the ADP-ribose transferase activity of the toxin-enzyme showed that there is no pre-steady-state complex formed during the catalytic cycle. Binding of NAD+ and smaller compounds representing the various parts of this substrate were investigated by the fluorescence quenching of the intrinsic toxin fluorescence. The binding data revealed a significant structural change in the enzyme upon NAD+ binding that could not be accounted for on the basis of the sum of the structural changes induced by the various NAD+ constituents. Product inhibition studies were conducted with nicotinamide and eEF-2-ADP-ribose, and the results indicate that the reaction involves a random-order ternary complex mechanism. Detailed kinetic analysis revealed that the eEF-2 substrate shows sigmoidal kinetic behavior with the enzyme, and fluorescence resonance energy transfer measurements indicated that wheat germ eEF-2 is oligomeric in solution.
authors.affiliation: Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Ontario N1G 2W1 Canada.
authors.forename: Souzan
authors.initials: S
authors.lastname: Armstrong
authors.forename: A Rod
authors.initials: AR
authors.lastname: Merrill
mesh_terms: D015057:1-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000244:Adenosine Diphosphate / Q000737:chemistry; D000246:Adenosine Diphosphate Ribose / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005098:Exotoxins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D053644:Naphthalimides; D009536:Niacinamide / Q000737:chemistry; D020652:Peptide Elongation Factor 2 / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D015363:Quinolones / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D037521:Virulence Factors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D015057:1-Naphthylamine; D036002:ADP Ribose Transferases; D000244:Adenosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D000249:Adenosine Monophosphate; D001427:Bacterial Toxins; D005098:Exotoxins; D009243:NAD; D053644:Naphthalimides; D009536:Niacinamide; D020652:Peptide Elongation Factor 2; D015363:Quinolones; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C086538:4-amino-1,8-naphthalimide