Table I.

Kinetic parameters of eCBL and site-directed mutants^a

Enzyme	kcat (s−1)	(mM)	(M−1s−1)
eCBL	34.1 ± 0.6	0.18 ± 0.01	(1.9 ± 0.1) × 105
R58A	9.7 ± 0.3	5.1 ± 0.3	(1.92 ± 0.05) × 103
R58Kb	n.s.	n.s.	385 ± 1
R59A	45.8 ± 0.4	1.02 ± 0.03	(4.5 ± 0.1) × 104
R59K	36.7 ± 0.8	1.51 ± 0.09	(2.4 ± 0.1) × 104
D116A	41.9 ± 0.7	0.67 ± 0.04	(6.2 ± 0.3) × 104
D116N	37.9 ± 0.4	0.55 ± 0.02	(6.9 ± 0.2) × 104
W340F	79 ± 1	1.35 ± 0.05	(5.9 ± 0.2) × 104
R372Ab	n.s.	n.s.	0.09 ± 0.04
R372K	8.1 ± 0.8	15 ± 2	550 ± 20
R372Lb	n.s.	n.s	0.23 ± 0.03

^aKinetic parameters reported are for hydrolysis of l-Cth. Reaction conditions: 2 mM DTNB, 0.01–6.4 mM l-Cth and 0.068–6.6 μM wild-type or mutant eCBL, depending on the activity of the enzyme, in assay buffer at 25°C. The data were fit to the Michaelis-Menten equation to obtain k_cat and and equation 1 to obtain .

^bn.s. indicates that exceeds the solubility limit of the l-Cth, such that was determined via linear regression.