Table 1.

The kinetic constants k_obs and k_obs/K^sto for cleavage of the various substrates by wild type Eco RPR

Substrate/[Mg2+]	kobs (min−1)	kobs/Ksto (min−1 µM−1)	Kd (µM)
pMini3bpUG
160 mM	0.86 ± 0.12	0.31 ± 0.12	2.8
800 mM	4.2 ± 0.48	1.6 ± 0.5	2.6
pMini3bpAbasicG
800 mM
+1	0.0010 ± 0.000006	0.00038 ± 0.000053	2.6
−1	0.0012 ± 0.00004	0.0010 ± 0.00014	1.2
pMini3bpCG
160 mM	0.0065 ± 0.0005	0.0006 ± 0.00012	11
800 mM	0.6 ± 0.3	0.19 ± 0.12	3.2
pMini3bpUA
160 mM	0.8 ± 0.06	0.11 ± 0.01	7.3
800 mM	4.8 ± 0.95	3.7 ± 0.4	1.3
pMini3bpCA
160 mM	0.29 ± 0.033	0.095 ± 0.015	3.1
800 mM	1.6 ± 0.2	1.3 ± 0.2	1.2
pMini3bpIsoCG
160 mM	0.0046 ± 0.0003	0.0013 ± 0.0003	3.5
800 mM	0.062 ± 0.014	0.009 ± 0.001	6.9
pMini3bpAG
800 mM	0.11 ± 0.061	0.034 ± 0.01	3.2
pMini3bpGG
800 mM	0.0088 ± 0.0036	0.0012 ± 0.0002	7.3
pMini3bpInoG
800 mM	0.22 ± 0.056	0.099 ± 0.026	2.2
pMini3bp2APG
800 mM	0.016 ± 0.007	0.0088 ± 0.003	1.8
pMini3bpDAPG
800 mM	0.025 ± 0.0048	0.013 ± 0.002	1.9
pMini3bpPuG
800 mM	0.029 ± 0.0094	0.023 ± 0.007	1.3

The experiments were performed under single-turnover conditions at different Mg²⁺ concentrations at pH 6.1 as described in ‘Materials and Methods’. The final concentration of substrate was >10 nM. The concentration of Eco RPR was varied between 0.8 and 29 µM dependent on the substrate. For details regarding the calculation of K_d, see the main text. The data represent mean and experimental errors calculated from at least three independent experiments.

ND = not determined.