Figure 3.

Attribution of phase 2 to a conformational transition. (a) Effect of saccharose (0% black, 10% red, 20% green, and 30% blue) on the reaction of Tsa1 (5 μM) with H₂O₂ (10 μM) monitored as in Figure 2, fitted against a three-exponential equation (red lines). Inset: effect of saccharose concentration on rate constants k₁^obs, k₂, and k₃ normalized to 0% saccharose. The stopped-flow mixer efficiency in viscous solutions was established by mixing Trp and up to 30% saccharose, which showed no artifactual effects on the dilution kinetics (Figure S2). (b) Near-UV circular dichroism spectra of 50 μM wild-type Tsa1 (plain) and Tsa1^Y190G F191G (dashed line) under the reduced (black) and disulfide (red) forms. Measurements were performed in a 1 cm cuvette in a phosphate 10 mM and NaF 100 mM buffer (pH 7) and are the average of three records. (c) Pre-steady-state kinetics for the reaction of Tsa1 (5 μM) with H₂O₂ (5, 10, 25, 50, 100, 200, 400, and 800 μM, light gray to black) monitored as in Figure 2, fitted against a biexponential equation (red lines). Inset: second-order plots and linear fits of the observed rate constants for the fast phase k₁^obs (circles, black line) and slow phase k₃ (diamond, green line). (d) Pre-steady-state kinetics for the reaction of Tsa1 (50 μM) with H₂O₂ (100 μM) monitored by a near-UV CD signal at 270 nm. The trace is the average of 50 runs, and the first-order fit is shown in red. (e) Effect of saccharose (0% black, 15% red, and 30% blue) on the reaction of human Prx1 (5 μM) with H₂O₂ (10 μM) monitored as in Figure 2 and fitted against a biexponential equation (red or black lines). Inset: effect of saccharose concentration on the rate constant of the increasing phase normalized to 0% saccharose.