PERTUSSIS TOXIN - TRANSITION-STATE ANALYSIS FOR ADP-RIBOSYLATION OF G-PROTEIN PEPTIDE ALPHA(I3)C20

Pertussis toxin from Bordetella pertussis is one of the ADP-ribosylati ng toxins which are the cytotoxic agents of several infectious disease s. Transition state analogues of these enzymes are expected to be pote nt inhibitors and may be useful in therapy. Pertussis toxin catalyzes the ADP-ribosylation of a cysteine in the synthetic peptide alpha(i3)C 20, corresponding to the C-terminal 20 amino acids of the ol-subunits of the G-protein G(i3). A family of kinetic isotope effects was determ ined for the ADP-ribosylation reaction, using H-3-, C-14 (-) and N-15- labeled NAD(+) as substrate. Primary kinetic isotope effects were 1.05 0 +/- 0.006 for [1'(N)-C-14] and 1.021 +/- 0.002 for [1(N)-N-15], the double primary effect of [1'(N)-C-14,1(N)-N-15] was 1.064 +/- 0.002. S econdary kinetic isotope effects were 1.208 +/- 0.014 for [1'(N)-H-3], 1.104 +/- 0.010 for [2'(N)-H-3], 0.989 1:+/- 0.001 for [4'(N)-H-3], a nd 1.014 +/- 0.002 for [5'(N)-H-3]. Isotope trapping experiments yield ed a commitment factor of 0.01, demonstrating that the observed isotop e effects are near intrinsic. Solvent D2O kinetic isotope effects are inverse, consistent with deprotonation of the attacking Cys prior to t ransition state formation. The transition state structure was determin ed by a normal mode bond vibrational analysis. The transition state is characterized by a nicotinamide leaving group bond order of 0.14, cor responding to a bond length of 2.06 Angstrom. The incoming thiolate nu cleophile has a bond order of 0.11, corresponding to 2.47 Angstrom. Th e ribose ring has strong oxocarbenium ion character, Pertussis toxin a lso catalyzes the slow hydrolysis of NAD(+) in the absence of peptides . Comparison of the transition states for NAD(+) hydrolysis and for AD P-ribosylation of peptide alpha(i3)C20 indicates that the sulfur nucle ophile from the peptide Cys participates more actively as a nucleophil e in the reaction than does water in the hydrolytic reaction. Particip ation of the thiolate anion al the transition state provides partial n eutralization of the cationic charge which normally develops at the tr ansition states of N-ribohydrolases and transferases. Thus, the presen ce of the peptide provides increased S(N)2 character in a loose transi tion state which retains oxocarbenium character in the ribose.