TABLE 1.

Constants for the binding of active site-directed fluorescent probes to thrombin and meizothrombin variants

Constants ± 95% confidence limits were determined from global fitting according to Scheme 1 (for thrombin (IIa)) or Scheme 2 (for mIIa). The symbolic constants correspond to those listed in the two schemes.

Species	Probe	k+1	k−1	fea	fE	k+2	k−2	KE,Pb	k+3	k−3	Ke,P	KConf
		s−1	s−1			μm−1·s−1	s−1	nm	μm−1·s−1	s−1	nm
IIa	I-2581	98.9 ± 2.8	20.5 ± 1.6	0.18	0.82	49.4 ± 0.6	1.02 ± 0.001	20.6 ± 0.02	NAc	NA	NA	NA
IIaA195	I-2581	81.4 ± 2.1	16.8 ± 0.9	0.17	0.83	34.2 ± 0.3	5.84 ± 0.04	171 ± 3	NA	NA	NA	NA
IIa	DAPA	113 ± 2	27.1 ± 0.6	0.19	0.81	71.1 ± 0.6	0.23 ± 0.01	3.2 ± 0.11	NA	NA	NA	NA
mIIaA195	I-2581	1.67 ± 0.01	2.33 ± 0.06	0.58	0.42	32.7 ± 0.3	5.95 ± 0.01	182 ± 1.3	0.95 ± 0.11	0.89 ± 0.01	940 ± 10	0.27 ± 0.09
mIIaQQQ	I-2581	1.67 ± 0.01	1.93 ± 0.02	0.54	0.46	57.3 ± 0.2	0.54 ± 0.01	9.3 ± 0.01	1.76 ± 0.01	2.30 ± 0.23	1310 ± 110	0.008 ± 0.001
mIIaQQQ	DAPA	1.70 ± 0.17	1.89 ± 0.08	0.53	0.47	92.5 ± 0.6	0.26 ± 0.006	2.84 ± 0.05	2.33 ± 0.07	0.58 ± 0.06	250 ± 20	0.013 ± 0.003

^a Fraction of enzyme in the e and E forms in the initial equilibrium is denoted by fe and fE.

^b K_E,P = k₋₂/k₊₂ is the equilibrium dissociation constant for P binding to E. K_e,P = k₋₃/k₊₃ is the equilibrium dissociation constant for P binding to e. Propagation of errors in the rate constants was used to estimate confidence limits in the calculated equilibrium constants (59).

^c NA, not applicable.