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. 2008 Jun 27;9(8):802–809. doi: 10.1038/embor.2008.90

Table 1.

Relative ADP-ribosyltransferase, glycohydrolysis and NAD+-binding activity of Pseudomonas aeruginosa ExoAc wild type and L3 mutants

ExoAc Relative kcat (ADPRT)* Relative kcat (GH)‡ Relative KD (NAD+)§
Wt 1.00±0.09 1.00±0.02 1.00±0.09
E546Aa 0.0012±0.00002 0.24±0.008 1.77±0.34
E547Aa 0.795±0.077 0.83±0.14 1.97±0.77
E548Aa 1.669±0.298 0.76±0.01 2.69±0.11
G549Aa 0.770±0.076 0.30±0.005 8.00±0.94
G550Aa 0.562±0.166 0.41±0.04 6.60±0.54
R551Aa 0.380±0.024 0.32±0.005 2.69±0.03
L552Aa 2.365±0.527 0.58±0.002 4.43±0.57
E553Aa 0.0015±0.00005 0.07±0.009 2.57±0.86
E546Db 0.00025±0.00005 0.45±0.002 3.83±0.89
E546Fd 0.007±0.0002 0.69±0.05 1.97±0.11
E546Hb 0.0012±0.0002 0.59±0.009 1.03±0.09
E546Nb 0.0010±0.00009 0.67±0.03 2.14±0.26
E546Qb 0.011±0.002 0.80±0.04 2.31±0.71
R551Eb 0.011±0.005 0.09±0.001 5.03±0.51
R551Hb ≈0∣∣ 0.41±0.002 2.29±0.46
R551Kb 0.317±0.024 0.69±0.03 1.29±0.03
R551Qb 0.185±0.014 0.38±0.01 2.63±0.39
R551Cb ≈0∣∣ 0.10±0.006 5.57±0.91
E546A/R551Ac 0.0001±0.0001 0.84±0.04 1.11±0.03
E546D/R551Kc 0.0009±0.0013 0.42±0.0002 2.71±0.74
E546R/R551Ec 0.00001±0.000002 0.38±0.01 1.29±0.49
ADPRT, ADP-ribosyltransferase; ExoA, exotoxin A; GH, glycohydrolysis; Wt, wild type. *The relative ADPRT activity was set at 1.00 for the wild-type ExoAc enzyme (a746±18 min−1; b628±8 min−1; c847±21 min−1; d900±20 min−1). Briefly, various concentrations of toxin (5–500 nM) were mixed with 12 μM elongation factor 2 (eEF2) and different amounts of ɛ-NAD substrate in 20 mM Tris–HCl buffer, pH 7.9, at 25°C (70 μl) and the fluorescence time-based data were collected for 300 s. ‡The relative GH activity was measured as described above except without eEF2 and the time course was for a period of 60 min. The relative GH activity was set at 1.00 for the wild-type ExoAc and ranged from 0.062 to 0.13 min−1. §The NAD+-binding ability of wild-type and mutant ExoAc proteins was measured by the quenching of the intrinsic Trp fluorescence caused by the binding of NAD+ to the active site. In this case, the toxin concentration was 1.5 μM. The KD of wild-type protein was 35±3 μM. ∣∣The ADPRT activity could not be accurately measured as it was nearly zero and close to the background signal. The kinetic and equilibrium binding data represent the mean±s.d. from four independent experiments.