Table 5.

Kinetic Parameters for GS–EA Binding to rGSTA1-1 WT and Site-Directed Mutants at 15 °C

	WT	F220Y	F220I	F10A	F10Y	F10L
amp1a	0.0091	0.0029	0.0074	0.013		0.0045
k1 (μM−1 s−1)b,c	6.11	7.92	4.81	0.41		0.65
amp−1	0.0043	0.0044	0.0037	0.0098		0.0077
k−1(exp) (s−1)d	15.9	9.37	20.44	8.04		13.3
k−1(calc) (s−1)c	14.7	6.84	24.22	9.81		14.1
K1 (μM−1)e	2.60	1.18	4.25	19.6	2.03	22.1
amp2	0.051	0.082	0.032	0.046	0.034	0.025
k2 (s−1)c	17.6	29.6	10.2	0.811	26.3	1.28
amp−2	0.018	0.022	0.021	0.013	0.014	0.011
k−2(exp) (s−1)d	1.51	0.77	4.26	0.59	8.1	0.48
k−2(calc) (s−1)c	1.89	0.83	6.96	0.51	11.9	0.51
K2f	0.086	0.026	0.42	0.73	0.026	0.42
Kd (μM)g	0.19	0.03	1.25	8.25	0.48	6.05

^aAmplitude values (amp₁, amp₋₁, amp₂, and amp₋₂) refer to the magnitude of the preexponential terms for the rate constants k₁, k_−1(exp), k₂, and k_−2(exp), as determined from stopped-flow experiments.

^bThe rate constants for all enzymes were determined from duplicate or triplicate experiments, where 15–30 scans were averaged. The standard deviation was less than ±0.8 for k₁, ±0.7 for k₋₁, ±0.9 for k₂, and ±0.5 for k₋₂.

^cKinetic binding and dissociation constants were determined by curve fitting the k_obs versus GS–EA plots to eqs 3 and 4.

^dRates of GS–EA dissociation were determined directly from a fit of the raw data to a single (1) or double (2) exponential equation.

^eK₁ is determined from the ratio k_−1(exp)/k₁ or eq 5.

^fK₂ is determined from the ratio k_−2(exp)/k₂.

^gEquilibrium dissociation constants were determined from K_d(calc) = K₁[k₋₂/(k₂ + k₋₂)].