4 muh

Enzyme activity of Pks13-TE
was assessed using 4-methylumbelliferyl heptanoate (4-MUH, Sigma)
as a fluorogenic substrate in a 96-well plate format. To make initial
velocity measurements, Pks13-TE (0.5 μM) in 0.1 M Tris-HCl,
pH 7 buffer was incubated with different concentrations of 4-MUH (0.2–200
μM in DMSO, 1% DMSO final) in a 100 μL reaction volume,
and the fluorescence of the hydrolyzed product 4-methylumbelliferone
was read (excitation at 355 nm and emission at 460 nm) using a PolarStar
Omega plate reader (BMG Labtech) at 5–10 min intervals over
120–140 min. The reaction rate was observed to be linear in
the measured range. 4-MUH in buffer alone was included as a control
to quantify its background hydrolysis. Data points were plotted as
an average of duplicates and analyzed using Prism software (GraphPad)
to determine the kinetic parameters K_m, V_max, and k_cat. The experiment was repeated in 20 mM HEPES, 134 mM potassium acetate,
8 mM sodium acetate, 4 mM sodium chloride, and 0.8 mM magnesium acetate,
pH 7.2.

Activity of Pks13-TE was assessed using 4-methylumbelliferyl heptanoate (4-MUH, Sigma) as a fluorogenic substrate in a 96-well plate format (Richardson and Smith, 2007 (link)). To make initial velocity measurements, Pks13-TE (1 μM) in 0.1 M Tris-HCl, pH 7 buffer was incubated with different concentrations of 4-MUH (2-150 μM in DMSO) in a 100 μL reaction volume, and the fluorescence of the hydrolyzed product 4-methylumbelliferone was read (excitation at 355 nm and emission at 460 nm) (PolarStar Omega plate reader BMG Labtech) at 5-10 min intervals over 80-120 min. The reaction rate was observed to be linear in the measured range. 4-MUH in buffer alone was included as a control to quantify its background hydrolysis. Data points were plotted as an average of triplicates and each experiment was repeated twice independently. The initial velocity data was curve fit to Michaelis-Menten equation by nonlinear regression using Prism software (GraphPad) to determine the kinetic parameters K_m and V_max. The assay and data analysis for Pks13-TE mutants was done the same way as that for the wild-type protein with the 4-MUH concentration varying from 2 to 300 μM.