Figure 3.

Enzyme kinetics and inhibition constant. (A, B) Michaelis-Menten Kinetics for the forward reaction of rBoLDH. Variations of rBoLDH activity with concentration of pyruvate and NADH. (C, D) Michaelis-Menten Kinetics for the reverse reaction of rBoLDH. Variations of rBoLDH activity with concentration of lactate and NAD⁺. (E, F) Michaelis-Menten Kinetics for the reverse reaction of rBoLDH. Variations of rBoLDH activity with concentration of lactate and APAD⁺. (G) Michaelis-Menten-based kinetics of inhibition of rBoLDH activity in the presence of 5 µM gossypol. The changes of density of NAD⁺ were monitored at OD₃₄₀. (H) Enzyme activity test for two variants of rBoLDH. The forward assays were performed at 25°C in pH 7.5 50 mM Tris-HCl buffer, containing 50 ng rBoLDH, 2 mM pyruvate and 0.8 mM NADH. S, substrate as indicated; V_o, initial velocity. The error bar represents mean ± SD (n=3), and Non-linear regressions and XY graph were drawn with GraphPad5.0.