Table 5.
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 |
Amplitude 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.
The 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.
Kinetic binding and dissociation constants were determined by curve fitting the kobs versus GS–EA plots to eqs 3 and 4.
Rates of GS–EA dissociation were determined directly from a fit of the raw data to a single (1) or double (2) exponential equation.
K1 is determined from the ratio k−1(exp)/k1 or eq 5.
K2 is determined from the ratio k−2(exp)/k2.
Equilibrium dissociation constants were determined from Kd(calc) = K1[k−2/(k2 + k−2)].