Kinetic constants for WT GlTIM and the C222 mutants.

n.a. not available; experimental data could not be fitted.
Initial velocity rates at GAP concentrations ranging from 0.3 to 3 mM were fitted to the Michaelis-Menten equation to obtain Vmax and Km. The kcat values were calculated from Vmax considering a molecular mass of 27.7 kDa. The Ki values for 2-PG were calculated from inhibition assays; initial velocity rates from 0.3 to 3 mM at fixed variable concentrations of 2-PG were globally fitted to a simple competitive inhibition model. Kd values for 2-PG were obtained from the change in the intrinsic fluorescence of GlTIM in response to the addition of this ligand as indicated in Material and Methods. For all experiments, the calculated values are the average (± standard error) of two independent experiments.