Fig. 4
The substrate-inhibition effect was relieved by LDHB-S162D. a The dissociation constant of NADH for LDH was assessed by isothermal titration calorimetry (ITC) assay. ITC results were shown for interactions of NADH with LDHB, LDHB-S162D, and LDHA. The Kd values were labeled at the bottom. b ITC results were shown for oxamate interactions with LDHB-NADH complex, LDHB-S162D-NADH complex and LDHA-NADH complex. The Kd values were labeled at the bottom. c The catalytic activity (Pyruvate to lactate) of LDHA, LDHB, and LDHB S162D were measured at different concentrations of pyruvate. The relative enzyme activity for LDHB, LDHB-S162D, and LDHA were plotted against the concentrations of pyruvate (logarithm of 2). d The catalytic rates of the forward reactions for LDHA, LDHB and LDHB S162D were plotted over physiological concentrations of pyruvate. Student t-test were performed between the rate of LDHB and LDHB S162D at 0.5 and 1.25 mM pyruvate. Curve fitting was conducted using Prism software. The error bar in panels a, b, c, d represents the standard error of mean(SEM), n = 6 independent experiments for LDHB, 7 for LDHB-S162D, 4 for LDHA in panels a, n = 4 independent experiments for LDHB, 6 for LDHB-S162D, 4 for LDHA in panels b, n = 4 independent experiments for LDHB, 3 for LDHB-S162D, 4 for LDHA in panels c and d. Source data are provided as a Source Data files. (Student’s t-test *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant).
