Table 2. Substrate-specific activity of the engineered enzymes compared to the wild-type.

The substrates used were 3 mM CDNB, 1 mM DCNB, 0.1 mM PNPBr, 0.1 mM PNBC and 0.2 mM EA. The reactions were performed at a constant GSH concentration (appropriate for each enzyme). ^aSome of these values have been previously reported and are shown for purposes of comparison [19,20]. One-way ANOVA with Dunnett's multiple comparison test was performed with wild-type as control; statistical significance is shown by † for P<0.05, ‡ for P<0.01 and § for P<0.001.

	Specific activity (μmol·min−1·mg−1)
Enzyme	CDNB	DCNB	EA	PNPBr	PNBC
Wild-typea	52.50±0.52	0.035±0.006	0.286±0.062	0.074±0.012	0.064±0.002
H38Aa	21.70±0.20§	0.037±0.001	0.146±0.016§	0.040±0.004§	0.013±0.002§
H38Ea	11.21±0.24§	<0.001	0.191±0.026§	<0.008	<0.005
H38Fa	3.76±0.09§	<0.001	0.230±0.019†	<0.005	<0.003
H38D	2.33±0.07§	<0.001	0.111±0.011§	<0.001	<0.003
H38K	2.02±0.07§	<0.001	0.165±0.007§	<0.003	<0.002
M39A	18.84±0.66§	0.012±0.001§	0.183±0.011§	<0.005	<0.003
M39F	28.67±0.71§	0.043±0.002†	0.166±0.010§	0.029±0.008§	0.065±0.009
N47A	0.13±0.01§	<0.001	0.079±0.001§	<0.001	<0.001
Q49A	33.36±0.92§	0.023±0.002‡	0.244±0.012	0.050±0.002§	0.044±0.001‡
Q49E	41.76±0.60§	0.032±0.001	0.244±0.020	0.020±0.001§	0.048±0.003‡
H50Aa	3.80±0.10§	0.015±0.007§	0.146±0.050§	0.012±0.002§	0.016±0.007§
H50Ea	0.15±0.01§	<0.001	0.026±0.004§	<0.005	<0.005
H50Ya	0.47±0.01§	<0.001	0.064±0.008§	<0.008	<0.005
H50K	6.28±0.04§	0.007±0.001§	0.053±0.020§	<0.007	<0.004
H50F	0.93±0.03§	<0.001	0.077±0.005§	<0.001	<0.001
C51A	15.44±0.58§	0.009±0.001§	0.046±0.001§	0.019±0.001§	<0.001
C51D	1.24±0.03§	<0.001	0.080±0.004§	<0.002	<0.001