. Author manuscript; available in PMC: 2013 Apr 3.

Published in final edited form as: Biochemistry. 2012 Mar 21;51(13):2940–2949. doi: 10.1021/bi3001903

Table 1.

Thermodynamic and kinetic parameters for wild-type and mutant variants of vTopo^a

Construct	K_d (nM)	K_cl	k_cl (min⁻¹)	k_off (min⁻¹)	k_on (M⁻¹min⁻¹) × 10⁻²	τ0.18 (s)	ME^I	ME^P
WT	139 ± 55	0.25 ± 0.03	0.14 ± 0.01	0.104 ± 0.003	3.12 ± 0.04	3	1.00	1.00
K35A	132 ± 35	0.32 ± 0.03	0.05 ± 0.001	0.093 ± 0.013	2.11 ± 0.13	5	0.41 ± 0.33	1.07 ± 0.04
K35E	272 ± 88	0.16 ± 0.02	0.016 ± 0.004	0.142 ± 0.002	2.70 ± 0.20	10	1.34 ± 0.42	0.96 ± 0.05
R67A	148 ± 80	0.03 ± 0.01	0.005 ± 0.001	0.147 ± 0.013	2.75 ± 0.09	22	0.34 ± 0.81	1.08 ± 0.10
R67E	ND	ND	0.004 ± 0.001	ND	ND	255	0.94 ± 0.43	1.01 ± 0.05
K271A	282 ± 118	0.09 ± 0.02	0.010 ± 0.0002	0.030 ± 0.015	3.44 ± 0.04	12	−1.04 ± 0.67	1.25 ± 0.08
K271E	ND	ND	0.006 ± 0.0004	0.006 ± 0.0003	2.41 ± 0.08	10	−2.23 ± 0.36	1.39 ± 0.04

K_d is the dissociation constant for noncovalent DNA binding (Scheme 1), K_cl = k_cl/k_r is the equilibrium constant for reversible strand cleavage (Scheme 1), k_cl is the rate constant for single turnover irreversible strand cleavage, k_off and k_on are the rate constants for 12mer strand dissociation and association (eq 3), τ_0.18 is the time for 18% substrate reaction in the supercoil unwinding assay, ME^I is the mutational effect on processivity based on intermediate topoisomer accumulation (eq 5), and ME^P is the mutational effect on processivity based on product accumulation (eq 5).