Table 3.

Steady-state kinetic parameters for wild-type and mutant forms of Abl

Construct	KDATP, μM	KDPeptide, μM	kcat, s−1	α	Calc. KmATP @ 2 mM peptide, mM	Calc. KmPeptide @ 2 mM ATP, mM
WT	69 ± 2	1,107 ± 57	19.0 ± 0.5	1.8 ± 0.2	97 ± 8	1,975 ± 236
F317L	76 ± 1	755 ± 11	25.6 ± 0.1	1.48 ± 0.02	100 ± 13	1,343 ± 176
G250E	47 ± 2	438 ± 43	24.0 ± 0.3	2.2 ± 0.3	96 ± 13	952 ± 157
Y253F	98 ± 5	1,164 ± 50	38 ± 2	1.4 ± 0.3	104 ± 14	1,617 ± 344

Measured and derived steady-state parameters. Shown are fits of activity assays with varying concentrations of both peptide substrate (0.25 to 2 mM) and ATP (25 μM to 2 mM) shown in Fig. 4 and SI Appendix, Fig. S6. Data are fitted to the general velocity equation for a sequential random Bi–Bi reaction (see also SI Appendix, Eq. 1). This yields catalytic activity (k_cat), binding affinity of ATP (K_D^ATP), and binding affinity of peptide substrate (K_D^Peptide), as well as proportionality factor α. α quantifies the degree that binding of substrate one either increases (a < 1) or decreases (a > 1) affinity of the other substrate to the enzyme. These parameters can then be used to calculate the K_m^ATP as well as K_m^Peptide at fixed concentrations of the other substrate (see also SI Appendix, Eqs. 2 and 3). Calc., calculated.