Toward the elucidation of the catalytic mechanism of the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A

Biochemistry. 2004 Jan 13;43(1):183-94. doi: 10.1021/bi034772u.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Naphthylamine / analogs & derivatives*
  • 1-Naphthylamine / chemistry
  • ADP Ribose Transferases / antagonists & inhibitors
  • ADP Ribose Transferases / chemistry*
  • ADP Ribose Transferases / genetics
  • Adenosine Diphosphate / chemistry
  • Adenosine Diphosphate Ribose / chemistry
  • Adenosine Monophosphate / chemistry
  • Bacterial Toxins / antagonists & inhibitors
  • Bacterial Toxins / chemistry*
  • Bacterial Toxins / genetics
  • Binding, Competitive / genetics
  • Catalysis
  • Enzyme Activation / genetics
  • Exotoxins / antagonists & inhibitors
  • Exotoxins / chemistry*
  • Exotoxins / genetics
  • Fluorescence Resonance Energy Transfer
  • Kinetics
  • Mutagenesis, Site-Directed
  • NAD / chemistry
  • Naphthalimides
  • Niacinamide / chemistry
  • Peptide Elongation Factor 2 / chemistry
  • Protein Binding / genetics
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / genetics
  • Quinolones / chemistry
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Substrate Specificity / genetics
  • Virulence Factors / antagonists & inhibitors
  • Virulence Factors / chemistry*
  • Virulence Factors / genetics

Substances

  • Bacterial Toxins
  • Exotoxins
  • Naphthalimides
  • Peptide Elongation Factor 2
  • Quinolones
  • Recombinant Proteins
  • Virulence Factors
  • NAD
  • 4-amino-1,8-naphthalimide
  • Adenosine Diphosphate Ribose
  • Niacinamide
  • Adenosine Monophosphate
  • Adenosine Diphosphate
  • 1-Naphthylamine
  • ADP Ribose Transferases
  • Pseudomonas aeruginosa exotoxin A