Bacterial protein toxins are the most powerful human
poisons known, exhibiting an LD(50) of 0.1-1 ng kg(-)(1). A major subset of such toxins is the
NAD(+)-dependent
ADP-ribosylating
exotoxins, which include
pertussis,
cholera, and
diphtheria toxin.
Diphtheria toxin catalyzes the ADP ribosylation of the
diphthamide residue of eukaryotic
elongation factor 2 (eEF-2). The transition state of ADP ribosylation catalyzed by
diphtheria toxin has been characterized by measuring a family of kinetic
isotope effects using (3)H-, (14)C-, and (15)N-labeled
NAD(+) with purified yeast eEF-2.
Isotope trapping experiments yield a commitment to catalysis of 0.24 at saturating eEF-2 concentrations, resulting in suppression of the intrinsic
isotope effects. Following correction for the commitment
factor, intrinsic primary kinetic
isotope effects of 1.055 +/- 0.003 and 1.022 +/- 0.004 were observed for [1(N)'-(14)C]- and [1(N)-(15)N]
NAD(+), respectively; the double primary
isotope effect was 1.066 +/- 0.004 for [1(N)'-(14)C, 1(N)-(15)N]
NAD(+). Secondary kinetic
isotope effects of 1.194 +/- 0.002, 1.101 +/- 0.003, 1.013 +/- 0.005, and 0.988 +/- 0.002 were determined for [1(N)'-(3)H]-, [2(N)'-(3)H]-, [4(N)'-(3)H]-, and [5(N)'-(3)H]
NAD(+), respectively. The transition state structure was modeled using density functional theory (B1LYP/6-31+G) as implemented in Gaussian 98, and theoretical kinetic
isotope effects were subsequently calculated using Isoeff 98. Constraints were varied in a systematic manner until the calculated kinetic
isotope effects matched the intrinsic
isotope effects. The transition state model most consistent with the intrinsic
isotope effects is characterized by the substantial loss in bond order of the
nicotinamide leaving group (bond order = 0.18, 1.99 A) and weak participation of the attacking
imidazole nucleophile (bond order = 0.03, 2.58 A). The transition state structure imparts strong oxacarbenium ion character to the
ribose ring even though significant bond order remains to the
nicotinamide leaving group. The transition state model presented here is asymmetric and consistent with a dissociative S(N)1 type mechanism in which attack of the
diphthamide nucleophile lags behind departure of the
nicotinamide.