Transition State Structure for the Hydrolysis of NAD Catalyzed by Diphtheria Toxin

Abstract

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 absence of its physiological acceptor, DTA catalyzes the slow hydrolysis of NAD(+) to ADP-ribose and nicotinamide, a reaction that can be exploited to measure kinetic isotope effects (KIEs) of isotopically labeled NAD(+)s. Competitive KIEs were measured by the radiolabel method for NAD(+) molecules labeled at the following positions: 1-(15)N = 1.030 ± 0.004, 1'-(14)C = 1.034 ± 0.004, (1-(15)N,1'-(14)C) = 1.062 ± 0.010, 1'-(3)H = 1.200 ± 0.005, 2'-(3)H = 1.142 ± 0.005, 4'-(3)H = 0.990 ± 0.002, 5'-(3)H = 1.032 ± 0.004, 4'-(18)O = 0.986 ± 0.003. The ring oxygen, 4'-(18)O, KIE was also measured by whole molecule mass spectrometry (0.991 ± 0.003) and found to be within experimental error of that measured by the radiolabel technique, giving an overall average of 0.988 ± 0.003. The transition state structure of NAD(+) hydrolysis was determined using a structure interpolation method to generate trial transition state structures and bond-energy/bond-order vibrational analysis 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 was 0.02 (bond length = 2.65 Å), while the bond order to the approaching nucleophile was 0.03 (2.46 Å). This is an A(N)D(N) mechanism, with both leaving group and nucleophilic participation in the reaction coordinate. Fitting the transition state structure into the active site cleft of the X-ray crystallographic structure of DTA highlighted the mechanisms of enzymatic stabilization of the transition state. Desolvation of the nicotinamide ring, stabilization of the oxocarbenium ion by apposition of the side chain carboxylate of Glu148 with the anomeric carbon of the ribosyl moiety, and the placement of the substrate phosphate near the positively charged side chain of His21 are all consistent with the transition state features from KIE analysis.

Figure 1

NAD⁺ molecule with positions of isotopic labels (³H, ¹⁴C, ¹⁵N, ¹⁸O) in bold type.

Figure 2

Reaction coordinate diagram. Positions in reaction coordinate space are illustrated by plotting the leaving group bond order (n_LG) on the ordinate and nucleophile bond order on the abscissa (n_Nu). Thus, the reaction proceeds from the substrates in the lower left corner to products in the upper right. A classical D_N + A_N (S_N1) reaction mechanism involves formation of an oxocarbenium ion intermediate, with complete loss of the leaving group, before the nucleophile bond order starts to increase. A concerted, synchronous A_ND_N (S_N2) mechanism follows the dotted diagonal line, where increase in n_Nu at each point matches exactly the loss of n_LG.

Figure 3

Distortional KIEs as modeled with EtOH. The bond angle, θ = ∠C2–C1–O1, is analogous to the bond angle ∠C4′–C5′–O5′ of NAD⁺ and the 1-³H KIE analogous to the 5′-³H KIE.

Figure 4

Hanes plot of kinetic constants for DTA-catalyzed NAD⁺ hydrolysis in 50 mM potassium phosphate (pH 6.0) at 37 °C.

Figure 5

Commitment to catalysis. The ordinate intercept, 358, is the reciprocal of the fraction of NAD⁺ that would react with DTA, without dissociation, at infinite concentration of NAD⁺. This corresponds to a commitment factor of 0.0028.

Figure 6

Match of predicted versus experimental KIEs. For each isotopic label, the colored area represents the match of the predicted with the experimental KIEs as a function of n_LG and n_Nu in this More–O'Ferrall-type reaction coordinate diagram. The light shading represents the 95% confidence interval of each measured KIE, with dark shading representing the exact measured KIE (approximately ± 0.001): blue, 1-¹⁵N; red, 1′-¹⁴C; gray, 1′-³H; green, 2′-³H; orange, 4′-¹⁸O. The predicted transition state structure is indicated by *. Only the top left (dissociative) corner of reaction coordinate space is shown.

Figure 7

Structures of reactant, transition state, and the hypothetical oxocarbenium ion, with independent, noninteracting nicotinamide and water molecules. (a) Atoms present in the cutoff models used in BEBOVIB calculations are colored by element; all others are colored gray. Changes in bond order between the reactant and the transition state are indicated. All atoms within two bonds of the isotopically labeled positions were included in KIE calculations, yielding “highly proper” cutoff models. (b) Electrostatic potentials projected onto the molecular surfaces of the full molecules in the same orientation as in part a, with red representing positive electrostatic potential and blue representing negative potential. Calculations were done at the RHF/6-31G** level. Molecular surfaces are at an electron density of 0.002 e/bohr³, and the electrostatic potential spectrum is from −0.1 (blue) to 0.1 hartree/e (red).

Figure 8

Stereodiagram of the transition state structure fitted into active site of DTA. Projection of the electrostatic potentials onto the molecular surface shows the complementarity of the contact site for the transition state. The ball-and-stick figure of the transition state on the lower right shows the orientation of the transition state in the active site. The ring oxygen, O4′, is labeled on the molecular surface. The region of positive potential (solid red) associated with C1′ of the TS is apposed with the negative potential (blue dots) of the carboxylate of Glu148. The negative electrostatic potential of the phosphate oxygens (solid blue) is near the positive potential (red dots) of His21 (not visible behind the phosphate). The proximity of the nucleophile water to the carboxylate of Glu148 raises the possibility that it promotes catalysis as a general base catalyst or by polarizing the H–O bond to enhance the nucleophilicity of the oxygen. The electrostatic potentials were calculated using the Delphi module of the program Insight II (Biosym Technologies, San Diego, CA). Charges were of 1+ were assigned to Lys, Arg, and His21 of DTA, 0.5+ to other His residues, and 1– to Glu and Asp. Point charges on the atoms of the transition state structure were assigned from the natural population analysis charges calculated from the wave function as in Figure 7. The molecular surfaces were calculated as the Connolly surfaces, but with the atomic radii reduced by a factor of 0.8 so that the surface approximates a smoothed van der Waals surface. The contact site of DTA includes all residues that contribute to the molecular surface surrounding the transition state: Tyr20, His21, Tyr54, Ser55, Thr56, Tyr65, Phe140, Glu148. Secondary structural elements of DTA, as calculated by the Kabsch and Sander criteria, are shown, with α-helices in purple and β-strands in blue. The transition state structure from Figure 7 was fitted into the active site cleft by allowing the bond angles between the nicotinamide and ribosyl moieties to vary, as well as the torsional angles about C4′–C5′ and C5′–O5′.