TRANSITION-STATE STRUCTURE FOR THE HYDROLYSIS OF NAD(-TOXIN() CATALYZED BY DIPHTHERIA)

Diphtheria toxin (DTA) uses NAD(+) as an ADP-ribose donor to catalyze the ADP-ribosylation of eukaryotic elongation factor 2. This inhibits protein biosynthesis and ultimately leads to cell death. In the absenc e of its physiological acceptor, DTA catalyzes the slow hydrolysis of NAD(+) to ADP-ribose and nicotinamide, a reaction that can be exploite d to measure kinetic isotope effects (KIEs) of isotopically labeled NA D(+)s. Competitive KIEs were measured by the radiolabel method for NAD (+) molecules labeled at the following positions: 1-N-15 = 1.030 +/- 0 .004, 1'-C-14 = 1.034 +/- 0.004, (1-N-15,1'-C-14) = 1.062 +/- 0.010, 1 '-H-3 = 1.200 +/- 0.005, 2'-H-3 = 1.142 +/- 0.005, 4'-H-3 = 0.990 +/- 0.002, 5'-H-3 = 1.032 +/- 0.004, 4'-O-18 = 0.986 +/- 0.003. The ring o xygen, 4'-O-18, KIE was also measured by whole molecule mass spectrome try (0.991 +/- 0.003) and found to be within experimental error of tha t measured by the radiolabel technique, giving an overall average of 0 .988 +/- 0.003. The transition state structure of NAD(+) hydrolysis wa s determined using a structure interpolation method to generate trial transition state structures and bond-energy/bond-order vibrational ana lysis to predict the KIEs of the trial structures. The predicted KIEs matched the experimental ones for a concerted, highly oxocarbenium ion -like transition state. The residual bond order to the leaving group w as 0.02 (bond length = 2.65 Angstrom), while the bond order to the app roaching nucleophile was 0.03 (2.46 Angstrom). This is an A(N)D(N) mec hanism, with both leaving group and nucleophilic participation in the reaction coordinate. Fitting the transition state structure into the a ctive site cleft of the X-ray crystallographic structure of DTA highli ghted the mechanisms of enzymatic stabilization of the transition stat e. Desolvation of the nicotinamide ring, stabilization of the oxocarbe nium ion by apposition of the side chain carboxylate of Glu148 with th e anomeric carbon of the ribosyl moiety, and the placement of the subs trate phosphate near the positively charged side chain of His21 are al l consistent with the transition state features from KIE analysis.