kcat/Km L-Histidine

Activity

Catalytic efficiency (kcat/Km)

1/M•s

Spectrophotometric/Colorimetric Assay

100 mM potassium phosphate

37 C

8.0

10 mU

340 nm

None

To measure steady-state kinetics for α-ketoglutarate, assays were performed by varying the α-ketoglutarate concentration up to 10 mM in the presence of 2 mM L leucine, 5 mM NAD+, 16 µM PLP, 1 U GDH, and approximately 10 mU of aminotransferase in 100 mM potassium phosphate buffer (pH 8, 37 °C). To measure steady-state kinetics for L histidine, L-leucine, and L-threonine, assays were performed by varying the amino-acid concentration up to 86 mM in the presence of 1.25 or 10 mM α-ketoglutarate, 5 mM NAD+, 16 µM PLP, 1 U GDH, and approximately 10 mU of aminotransferase in 100 mM potassium phosphate buffer (pH 8, 37 °C). The pH of all reaction mixtures were adjusted to 8.0 prior to initiation of the assay. Linear phases of the kinetic traces were used to measure initial reaction rates. Initial reaction rates at different amino-acid concentrations were fit to the Michaelis-Menten equation using Python ver.2.7.15 with the scipy.optimize.curve_fit function (scipy ver.1.1.0). For mutants displaying substrate inhibition, fitting of the kinetic data was done with a rate equation that takes into account this type of inhibition: v0 = (vmax[S])/(KM + [S] + [S]2/Ki).