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 ADPribose 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.

Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide 18O/16O measurements

“…The linearity of the data and the standard deviations of the replicates indicate that the precision of the isotope ratios obtained from the product ion should be sufficient to permit heavy atom isotope effects to be determined on nucleotides. Similar results were obtained for the protonated base product ion when [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine was analyzed (data not shown). The procedures described to obtain and analyze IDs by tandem MS for nucleotides (and peptides [22]) will permit heavy atom isotope effect studies to be conducted on these biomacromolecular substrates.…”

“…18 O 1 ]pGp were purified by HPLC (300 mm × 3.9 mm, 10 μm C 18 packing) eluted isocratically using 50 mM diisopropylethylamine (Aldrich), 1% acetic acid in water (pH 4.3) as the mobile phase. For [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine, samples were resolved by a gradient of acetonitrile in 0.2 M ammonium acetate (5% -20% acetonitrile in 60 min.). Appropriate fractions containing either uridine, UMP, TMP or pGp were collected.…”

“…The application of the analysis of isotope ratios by analysis of IDs was extended to tandemquadrupole time of flight mass spectrometry by analysis of [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine and UM[ 18 O 1 ]P with an Applied Biosystems Q-STAR. All analyses were performed by direct infusion of the nucleoside/nucleotide in 50% acetonitrile 1% formic acid.…”

“…IRMS can only analyze small, non-polar gasses such as CO 2 , N 2 , and H 2 ; thus, this technique can only be used when the atom of interest can be quantitatively converted to one of these gasses. Scintillation counting requires substantial double-labeling schemes involving specific isotopic pairs (usually 3 H and 14 C, although others can be used [10]), again limiting the molecules that can be analyzed.…”

“…Today, the development of gentler ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) allow larger molecules to be analyzed. Coupled with QMS, ESI makes possible the analysis of isotope ratios for numerous biologically relevant organic molecules with molecular masses in excess of 500 Da [13][14][15][16]. However, several design features prevent QMS from achieving the precision and accuracy obtained by IRMS.…”