Table II.

Kinetic parameters for inhibition of eCBL and site-directed mutants by AVG

Enzyme	IC50 (μM)a	Ki (μM)b	k2 (s−1)b	k1 (s−1)c
eCBL	1.7 ± 0.2	1.9 ± 0.6	(5 ± 1) × 10-4	263
R58A	1.74 ± 0.08	6 ± 1	(2.0 ± 0.4) × 10-3	333
R58K	23 ± 1	55 ± 7	(3.5 ± 0.3) × 10-3	64
R59A	1.34 ± 0.05	2.7 ± 0.9	(1.3 ± 0.4) × 10-3	481
R59K	4.6 ± 0.3	12 ± 3	(6 ± 1) × 10-4	50
D116A	2.6 ± 0.4	3.9 ± 0.9	(4.2 ± 0.9) × 10-4	108
D116N	3.3 ± 0.6	2.7 ± 0.8	(2.6 ± 0.8) × 10-4	92
W340F	43 ± 6	40 ± 10	(7 ± 2) × 10-4	17.5
R372A	n.d.	n.d.	n.d.
R372K	3500 ± 1400	9100 ± 400	(2.30 ± 0.02) × 10-3	0.25
R372L	n.d.	n.d.	n.d.

^aReaction conditions for IC₅₀ measurements: Enzyme (0.024–4.3 μM, depending on the activity of the mutant) was incubated with 0.05–10⁴ μM AVG in assay buffer at 25°C for 10 min. Activity was subsequently measured (n = 4) at a l-Cth substrate concentration of 0.1 mM and the data were fit to equation 3 to obtain the IC₅₀ value for each enzyme.

^bReaction conditions for measurement of K_i and k₂: Wild-type eCBL and site-directed mutants were incubated with 1.5 mM l-Cth and 0.005–7.5 mM AVG in assay buffer and the progress of the reactions was monitored for 30 min. The progress curves were fit to equation 5 to obtain k_obs values, which were plotted versus inhibitor concentration and fit to equation 6 to obtain the values of k₂ and K_i.

^cValues of the rate constant k₁, for the association of eCBL and AVG were calculated using the equation K_i = k₂/k₁.