Table 1.

Itk full-length	Vi/[Enzyme] (min−1)
Itk wild-type	0.81 ± 0.04
Itk K390R	0.001 ± 0.00
Itk F434A	0.003 ± 0.00
Itk F434G	0.19 ± 0.04
Itk L432I/F434A	0.60 ± 0.06
Itk C431V/F434A	0.009 ± 0.007
Itk L375V/F434A	0.021 ± 0.018
Itk H377V/F434A	0.030 ± 0.029
Itk isolated kinase domain
Background	0.004 ± 0.0005
Itk wild-type	0.004 ± 0.0003
Itk F434T	0.003 ± 0.0002
Itk F434I	0.005 ± 0.0001
Itk F434M	0.008 ± 0.0003
Itk L432I/F434M	0.010 ± 0.0006

Btk isolated kinase domain	(Without pY551 level normalization)	(pY551 level normalized)
Background	0.001 ± 0.000	0.001 ± 0.001
Btk wild-type	0.09 ± 0.001	0.10 ± 0.001
Btk T474A	0.06 ± 0.001	0.03 ± 0.015
Btk T474M	0.56 ± 0.060	0.53 ± 0.084
Btk T474I	0.06 ± 0.000	0.12 ± 0.008

Activity measurements were carried out at room temperature using 5 μCi of [³²P] ATP and Peptide B (aminohexanoyl biotin-EQEDEPEGIYGVLF-NH₂) as a substrate in a kinase assay buffer (50 mM Hepes pH 7.0, 10 mM MgCl₂, 1 mM DTT, 1 mg/mL BSA, and 1 mM Pefabloc) as described previously (10).