“…8,14) The amount of FAGLA hydrolyzed was evaluated using the molar absorption difference due to hydrolysis, Á" 345 ¼ À310 M À1 cm À1 , at 25 C. 8,14,28) The reaction was carried out in 40 mM acetate-NaOH buffer at pH 4.0-5.5, 40 mM MES-NaOH buffer at pH 5.5-7.0, 40 mM HEPES-NaOH buffer at pH 7.0-8.5, and TAPS-NaOH buffer at pH 8.0-9.0, each of which contained 10 mM CaCl 2 , at 25 C. Hydrolysis was carried out under pseudo first-order conditions, where the substrate concentration is much lower than the Michaelis constant (K m ) (>30 mM) 14) because of the slight solubility (<6 mM) of FAGLA. 8,14,28) Under the conditions, the kinetic parameters, K m and the molecular activity (k cat ), cannot be determined separately, and enzyme activity was evaluated by the specificity constant (k cat =K m ). The intrinsic k cat =K m , (ðk cat =K m Þ o ), and the proton dissociation constants (K e1 and K e2 ) for the bell-shaped pH-dependence of the activity (k cat =K m ) were calculated from eq.…”