TABLE 1.

Kinetic parameters at pH 6.5 for non-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
WT	75 ± 3.0	0.00028	75 ± 1.4	0.31 ± 0.03	1.00 ± 0.20
T67A	102 ± 3.4	0.00018	116 ± 2	0.25 ± 0.06	1.26 ± 0.19
R70Q	3.4 ± 0.14	0.0038	9.62 ± 0.49	4.10 ± 0.63	1.11 ± 0.38
R74Q	0.18 ± 0.014	0.03-0.09	0.20 ± 0.01	0.88 ± 0.11	0.90 ± 0.03
D90N	0.019 ± 0.0015d	0.04	0.03 ± 0.001	0.73 ± 0.14	1.62 ± 0.66
D94N	0.21 ± 0.014e	0.04	3.00 ± 0.48	52 ± 10	0.64 ± 0.10

^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 concentrations (2.5-60 mm), which are 10 to 20 times the K_m GSH for that enzyme. These concentrations were close to saturating for all the enzymes except D94N (for which 60 mm GSH was used).

^dA specific activity of 0.017 ± 0.0004 μmol/min/mg was measured using 0.15 mg of protein/ml in the assay.

^eA specific activity of 0.17 ± 0.015 μmol/min/mg was measured using 0.06 mg of protein/ml in the assay.