The complex impact of cancer-related missense mutations on the stability and on the biophysical and biochemical properties of MAPK1 and MAPK3 somatic variants

Table 5 Kinetic parameters and effect of temperature on kinase activity of MAPK1 variants wild type and variants^a

	K_m (μM)	kcat (s⁻¹)	[enzyme] (nM)	kcat/K_m (s⁻¹ μM⁻¹)	V_max (μM/sec)	T_max (°C)	E_a (kcal/mol)
Wild type	1.81 ± 0.29	2.20	5.0	1.22	1.10 × 10^–2	30.0	9.95 ± 0.41
GROUP I and II
E81K	2.64 ± 0.72	1.24	10.0	4.70 × 10^–1	1.24 × 10^–2	35.0	6.90 ± 0.95
R135K	6.19 ± 1.9	28.3	0.5	4.57	1.42 × 10^–2	35.0	7.02 ± 0.90
D162G	1.43 ± 0.47	9.30 × 10^–4	1437	6.50 × 10^–4	1.34 × 10^–3	40.0	6.94 ± 0.96
R191H	55	2.00 × 10^–2	2381	3.64 × 10^–4	4.83 × 10^–2	40.0	8.09 ± 0.99
Y316F	1.57 ± 0.3	6.83	1.0	4.35	6.83 × 10^–3	37.0	5.74 ± 0.26
P319S	4.26 ± 1.11	3.62 × 10^–1	100	8.50 × 10^–2	3.62 × 10^–2	35.0	7.56 ± 0.31
E322V	2.81 ± 0.68	3.80	5.0	1.35	1.90 × 10^–2	37.0	3.99 ± 0.26
GROUP III
E33Q	3.90 ± 0.67	1.98	5.0	5.08 × 10^–1	9.91 × 10^–3	25.0	11.1 ± 1.24
L121I	7.12 ± 2.09	7.20E-02	390.0	1.01 × 10^–2	2.80 × 10^–2	35.0	8.97 ± 1.40
L200F	3.23 ± 0.4	2.43E-02	500	7.52 × 10^–3	1.21 × 10^–2	30.0	5.76 ± 0.86
D235V	3.97 ± 0.67	3.10E-01	50	7.81 × 10^–2	1.55 × 10^–2	37.0	8.60 ± 0.88

^aThe catalytic activity of the P-MAPK1 wild type and variants was determined at the desired temperature with the substrate peptide AQT0490. Kinetic parameters were determined at 30 °C by using at least at 10 different concentrations of AQT0490. Ea was determined by Eq. (1) in the temperature range between 10 °C and the temperature of maximal activity (T_max) for each protein. Data are reported as the mean ± SE of the fit

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