A computational protocol to evaluate the effects of protein mutants in the kinase gatekeeper position on the binding of ATP substrate analogues

Table 1 Substrate phosphorylation by ATP, kcat/Km, IC₅₀ and predicted interaction energy for protein–ligand pairs

Kinases	Ligands	Experimental data	Predicted interaction energies (kcal/mol)
JNK kinase		% Substrate phosphorylation
JNK	N6-(benzyl)	99	0^a
	N6-(2-phenythyl)	98	0^a
	N6-(cyclopentyl)	97	0^a
	N6-(1-methylbutyl)	93	0^a
JNKM108GL168A	N6-(benzyl)	62	−17.34
	N6-(cyclopentyl)	59	−14.42
	N6-(1-methylbutyl)	47	−20.42
	N6-(2-phenythyl)	8	−33.0
v-Src tyrosine kinase		kcat/Km (min ⁻¹ M ⁻¹ )
v-Src	ATP	1.6*10⁵	−21.35
v-SrcI338A		1.4*10⁴	−19.91
v-SrcI338G		1*10⁴	−19.01
v-Src	N6-(benzyl) ATP	0	0^a
v-SrcI338A		2.5*10⁴	−14.92
v-SrcI338G		4.0*10⁴	−29.17
Tyrosine and serine/threonine kinases		IC ₅₀ (μM)
v-Src	PP1	5 ± 2	0^a
v-SrcI338F		8 ± 2	0^a
v-SrcI338 M		8 ± 1	0^a
v-SrcI338S		0.4 ± 0.05	−28.78
v-SrcI338T		0.1 ± 0.02	−33.32
v-SrcI338 V		0.1 ± 0.02	−27.41
v-SrcI338C		0.07 ± 0.02	−27.44
v-SrcI338A		0.005 ± 0.002	−39.26
v-SrcI338G		0.005 ± 0.002	−38.56
Fyn		0.05 ± 0.02	−36.81
FynT339A		0.005 ± 0.002	−36.21
Abl		0.3 ± 0.03	−32.93
AblT334A		0.03 ± 0.005	−33.86
CamKII		80 ± 10	0^a
CamKIIF89G		0.5 ± 0.1	−15.74
Cdk2		50 ± 10	0^a
Cdk2F80G		0.16 ± 0.03	−24.85
P38		0.82 ± 0.2	−34.61
P38T106A		0.0027 ± 0.005	−33.43
P38T106G		0.0027 ± 0.005	−32.78

ISSN: 1756-0500