Figure 4
Effects of oxamate on the catalytic reactions at pH 7.0. The reaction velocities for FNLDH (a), PALDH (b), and ECLDH (c) were measured in 50 mM MOPS-NaOH buffer (pH 7.0) containing 0.1 mM NADH, the indicated concentrations of pyruvate and several concentrations of oxamate. The concentrations of oxamate for FNLDH were 0 mM (open circles), 10 mM (closed circles), 20 mM (open triangles), 30 mM (closed triangles), and 40 mM (open squares). The concentrations of oxamate for PALDH were 0 mM (open circles), 0.1 mM (closed circles), 0.2 mM (open triangles), 0.3 mM (closed triangles), and 0.4 mM (open squares). The concentrations of oxamate for ECLDH were 0 mM (open circles), 5 mM (closed circles), 10 mM (open triangles), 15 mM (closed triangles), and 20 mM (open squares). The reaction velocity and the concentration of pyruvate were plotted reciprocally. The data for PALDH and ECLDH were interpreted using the equation for mixed type inhibition, whereas the data for FNLDH were interpreted using the equation for competitive type inhibition. The kinetic parameters were as follows; FNLDH: kcat = 79 ± 1 (s-1), Km= 0.39 ± 0.01 (mM), and Ki = 9.4 ± 0.2 (mM). PALDH: kcat = 410 ± 10 (s-1), Km = 0.074 ± 0.006 (mM), Ki = 0.29 ± 0.04 (mM), and Ki' = 0.33 ± 0.04 (mM). ECLDH: kcat = 670 ± 10 (s-1), Km = 5.6 ± 0.3 (mM), Ki = 20 ± 2 (mM), and Ki' ± 47 ± 6 (mM). GraFit ver 7.0.3 was used for non-linear regression and calculation of values. The lines were calculated with kinetic parameters.