TABLE 3.

Association (k_on) and dissociation (k_off) rate constants and apparent equilibrium dissociation (K_D) constants for the interaction of substrate plasminogen and μPG with wtSK/SK loop mutants complexed with immobilized HPG by SPR

Kinetic constants for the interaction of substrate HPG and μPG with wtSK/loop mutants: HPG (binary complex) were determined by global fitting to a 1:1 binding model, using the BIAcore 3000 evaluation software as described under “Experimental Procedures.” A stable binary complex between wtSK/SK loop mutants and HPG immobilized onto the SA chip was first made, and the binding of varying concentrations of substrate HPG (0.05–1 μm), or substrate μPG (1–6 μm) was monitored (also see supplemental Figs. S2 and S3 for the sensorgrams).

Ligand	Ligate HPG			Ligate μPG
	kon	koff	KD	kon	koff	KD
	m−1s−1 × 105	s−1 × 10−1	m × 10−6	m−1s−1 × 105	s−1 × 10−1	m × 10−6
wtSK·HPG	12 ± 1.5	1.8 ± 0.2	0.15 ± 0.03	1.4 ± 0.5	1.5 ± 0.20	1.1 ± 0.3
P177A·HPG	8.0 ± 1.8	1.6 ± 0.2	0.20 ± 0.04	1.5 ± 0.3	1.7 ± 0.15	1.1 ± 0.2
K180A·HPG	11 ± 2.0	1.5 ± 0.3	0.14 ± 0.02	1.1 ± 0.3	1.4 ± 0.15	1.2 ± 0.2
K180D·HPG	12.5 ± 2.0	1.5 ± 0.4	0.12 ± 0.02	1.2 ± 0.2	1.2 ± 0.15	1.0 ± 0.2