TABLE 2.

Kinetic parameters at pH 6.5 for His tag wild-type and mutant human GST π enzymes

Enzyme	Specific activitya	Protein concentration in assay	Vmaxb	Km GSHb	Km CDNBc
	μmol min−1 mg−1	mg/ml	μmol min−1 mg−1	mm	mm
His-WT	73.5 ± 2.5	0.0002	71 ± 1	0.32 ± 0.03	0.83 ± 0.09
His-T67A	102 ± 0.6	0.0004	101 ± 3	0.22 ± 0.04	0.90 ± 0.39
His-R70Q	6.2 ± 0.2	0.0066	11 ± 0.23	2.11 ± 0.18	0.87 ± 0.20
His-R74Q	0.19 ± 0.014	0.04	0.21 ± 0.006	0.41 ± 0.06	0.51 ± 0.08
His-D90N	0.02 ± 0.006d	0.04	0.02 ± 0.001	1.18 ± 0.26	0.85 ± 0.24
His-D94N	0.20 ± 0.018e	0.04	3.40 ± 0.83	61 ± 19	0.70 ± 0.17

^aThe standard assay solution contains 2.5 mm GSH, 3 mm CDNB, and 1 mm EDTA in 0.1 M potassium phosphate buffer (pH 6.5) in a total volume of 1 ml at 25 °C.

^bV_max and K_m values were determined by extrapolation to infinite concentration of the GSH, while maintaining (CDNB) at 3 mm in 0.1 M potassium phosphate buffer (pH 6.5) containing 1 mm EDTA using SigmaPlot for data analysis.

^cK_m values were determined by varying the concentration of CDNB (0.05-3 mm), while the GSH concentration was maintained at high concentrations (2.5-60 mm), which are close to saturating for all the enzymes except D94N.

^dA specific activity of 0.018 ± 0.0017 μmol/min/mg was measured using 0.37 mg of protein/ml in the assay.

^eA specific activity of 0.20 ± 0.008 μmol/min/mg was measured using 0.08 mg of protein/ml in the assay.