Steady-state kinetics

Activity

Catalytic efficiency (kcat/Km)

1/M•s

Spectrophotometric/Colorimetric Assay

100 mM sodium phosphate buffer supplemented with 100 mM sodium chloride

27

7.0

approximate values: 2 μM HG3, 50 nM HG3.3b, 10 nM HG3.7/HG3.14, 5 nM HG3.17/HG4

380 nm

None

All assays were carried out at 27 °C in 100 mM sodium phosphate buffer (pH 7.0) supplemented with 100 mM sodium chloride. Triplicate reactions with varying concentrations of 5-nitrobenzisoxazole (AstaTech) dissolved in methanol (10% final concentration) were initiated by addition of approximately 2 μM HG3, 50 nM HG3.3b, 10 nM HG3.7/HG3.14, or 5 nM HG3.17/HG4. Product formation was monitored spectrophotometrically at 380 nm (ε = 15,800 M−1 cm−1).5 Linear phases of the kinetic traces were used to measure initial reaction rates. Initial reaction rates at different substrate concentrations were fit to the Michaelis-Menten equation using GraphPad Prism.