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Oxidizing conditions, without DTT; reducing conditions, with 1 mM DTT. We estimated kcat, apparent KM for the substrate F6P and catalytic efficiency (kcat/KM) in a continuous spectrophotometric assay as detailed in Methods. In case of the oxidized wild-type enzyme and all variants except for Cys38Ser, we noticed a pronounced lag phase (kinetic hysteresis) that suggested a catalytic activation under turnover conditions. We thus provide both the steady-state activities after full activation as well as basal activities at t = 0 before activation. The x-fold change for kcat and KM for the reduced enzyme relative to the oxidized form at steady state is highlighted. All measurements were carried out in triplicate and are shown as mean ± s.d. The catalytic constant of NmTAL in the oxidized form represents an upper limit, as oxidized and reduced species cannot be quantitatively separated by chromatographic methods as in the case of NgTAL.
aForcatalytic activation phases, data were analysed as described in ‘Steady-state kinetic analysis’ in Methods.