kcat
Activity
Catalytic Rate Constant (kcat)
1/min
Spectrophotometric/Colorimetric Assay
50 mM HEPES,10 mM MgCl2, 2 mM DTT, 0.2% BSA, and 0.005% Tween 20
25 °C
7.5
Variant-dependent; refer to the details; 5-1500 nM
None
None
ATPase activity assays were as described previously (Her et al, Cell Chem Biol. 2016, 23:517-28) and performed in 384-well format using colorimetric phosphate detection (PiColorLock Gold, Novus) with a FlexStation 3 microplate reader (Molecular Devices). Reaction velocities at 200 μM ATP were initially measured at different enzyme concentrations to identify linear rates after 2 h at room temperature. ATP titration experiments were performed (1 mM maximum, 2-fold serial dilutions, and eight concentrations) with Km and kcat values calculated using the Michaelis-Menten equation.
Enzyme concentrations were as follows: 22 nM WT; 20 nM R95G; 12 nM R155H; 12.5 nM K615V; 15 nM N616F; 5 nM P472L; 6.25 nM Q473P; 6 nM V474A; 12 nM G481A; 30 nM N660K; 200 nM T688A; 3 nM A530T; 14 nM R567H; 8 nM L639F; 540 nM K251A; 21 nM E305Q; 1040 nM K524A; 95 nM E578Q; 10 nM K251A+P472L; 8 nM E305Q+P472L; 500 nM P472L+K524A; 115 nM P472L+E578Q; 1500 nM R635A; 700 nM R638A; 240 nM P472L+R635A; and 230 nM P472L+R638A.
Inhibitor profiling experiments were performed with 200 μM ATP, and compounds were serially diluted from the indicated maximum. Relative rates were fit to a 4-parameter inhibition dose-response equation using GraphPad Prism 7 to obtain IC50 values with standard error.