FIGURE 2.

Characterization of redox state dependent activity. A, AtKAT2 is inactive in the presence of CSSC and active in the presence of CSH. The rates of acetoacetyl-CoA cleaving by AtKAT2 were determined as a function of time and incubation with oxidant or reductant. 12.5 μm enzyme preparations were incubated at 25 °C in 35 mM Tris (pH 8.5), 0.1 m NaCl and 20 mm CSH (●), 10 mm CSSC (▾) or buffer (■). The effects of CSH (○) and CSSC (▿) on the substrate are included for reference. The incubations were sampled at the given time points by rapid mixing and 2500× dilution with 100 μm CoA, 50 μm acetoacetyl-CoA in a microcuvette with absorbance recorded at 233 nm. The final concentrations of AtKAT2, CSH, and CSSC in the reactions were 5, 8, and 4 nm, respectively. B, AtKAT2 changes tertiary structure upon incubation with CSSC. AtKAT2 was incubated with different ratios of [CSH]²/[CSSC] at 25 °C for 10 min, and tryptophan fluorescence was recorded at 333 nm with excitation wavelength 285 nm. The total concentration of CSH and CSSC was kept constant at 125 mg/liter. A two-state equation was fitted to the normalized (solid line) data and the midpoint of transition determined. C, Fluorescence spectra of reduced (solid line) and oxidized (dotted line) AtKAT2 at 1 and 0.03 m [CSH]²/[CSSC], respectively. Conditions as in B except emission recorded from 325 to 450 nm.