TABLE 1.

Summary of kinetic parameters

Maximum amount of binding (Δm_max), dissociation constant (K_d), binding (k_on), dissociation rate constants (k_off), and the cleavage rate constant (k_cat) of EcoRII and its derivatives for different DNAs. [DNA] = 19 ± 1 ng (0.55 ± 0.02 pmol) cm^–2 on a QCM, [EcoRII] = 1–30 nm, [EcoRII-N domain] = 1–500 nm, [K263A] = [R330A] = 1–5 nm, 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm DTT, [Mg²⁺ or Ca²⁺] = 5 mm, 25 °C. The obtained kinetic values contain ±5% experimental errors.

Run	Enzymes	DNA	Divalent cations	Δmmaxa	Kd	kond	koffd	kcat
				ng cm-2	nm	105m-1 s-1	10-3 s-1	10-3 s-1
1	EcoRII	2-site DNA	Mg2+	-	3.3b	3.0b	1.0b	18b
2		1-site DNA	Mg2+	46 (0.9)c	16a, 3.2d	4.8	1.6	0
3	EcoRII	2-site DNA	Ca2+	51 (1.0)c	5.6a, 2.0d	5.2	1.0	0
4		1-site DNA	Ca2+	51 (1.0)c	11a, 3.4d	5.0	1.7	0
5		0-site DNA	Ca2+	51 (1.0)c	71a, 77d	0.10	0.69	0
6		2-site digested DNA-α	Ca2+	∼15 (∼0.3)c	150a	-	-	-
7		2-site digested DNA-β	Ca2+	37 (0.7)c	13a	-	-	-
8	EcoRII N-domain	1-site DNA	Ca2+	14e (1.1)c, 32f (2.5)c	10e, 530f	1.4	2.2	-
9		0-site DNA	Ca2+	20 (1.6)c	130a, 122d	0.09	1.1	-
10	K263A	2-site DNA	Ca2+	-	3.7d	4.7	1.7	0
11		1-site DNA	Ca2+	-	3.4d	5.1	1.7	0
12	R330A	2-site DNA	Ca2+	-	3.3d	4.6	1.6	0
13		1-site DNA	Ca2+	-	3.1d	5.2	1.6	0

^aObtained from the saturation binding experiments according to Eq. 1.

^bObtained from curve fitting experiments according to Eqs. 7, 8, 9. K_d = k_off/k_on.

^cBinding ratio of EcoRII or the EcoRII N-domain to DNA.

^dObtained from curve fitting experiments according to Eqs. 3, 4, 5. K_d = k_off/k_on.

^e[EcoRII N-domain] = 1-10 nm.

^f[EcoRII N-domain] = 100-500 nm.