Figure 5
Effects of oxamate on the catalytic reactions at pH 8.0. a-c) The saturation curves for oxamate. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) containing 0.1 mM NADH, 2.5 mM (for FNLDH), 1.2 mM (for PALDH), or 7.5 mM (for ECLDH) pyruvate, and the indicated concentration of oxamate. The data for FNLDH, PALDH, and ECLDH were interpreted using the equation for competitive-type, mix-type, and noncompetitive-type inhibition of allosteric enzyme. The kinetic parameters were as follows; FNLDH: Ki = 51 ± 10 (mM), kcat, 80 ± 20 (s-1), nH' ± 1.1 ± 0.1, and Kact = 9.9 ± 3 (mM). PALDH: KiKi' / (Ki + Ki') = 2.0 ± 0.6 (mM), kcat' = 470 ± 100 (s-1), nH' = 1.7 ± 0.2, and Kact = 1.7 ± 0.4 (mM). ECLDH: Ki = 80 ± 3 (mM). The lines indicate the calculated saturation curves obtained with kinetic parameters. d-f) the saturation curves for pyruvate with or without oxamate. The reaction velocities for FNLDH (d), PALDH (e), and ECLDH (f) were measured in 50 mM Bicine-NaOH buffer (pH 8.0) in the presence of 0.1 mM NADH and the indicated concentrations of pyruvate with no effector (open circles), or 20 mM (for FNLDH), 2.5 mM (for PALDH), or 30 mM (for ECLDH) oxamate (closed circles). The lines indicate the calculated saturation curves obtained with kinetic parameters.