TABLE 2.

Kinetic constants for the catalytic reaction of furin and furin alanine loop mutants with two RXXR fluorogenic substrates

Kinetic constants were measured at pH 7.5, 25 °C from the dependence of initial rates of substrate hydrolysis on substrate concentration as described under “Experimental Procedures.” Errors represent S.E. from nonlinear regression fits of data by the Michaelis-Menten equation.

Furin surface loop variant	Furin residues mutated into alanines	Boc-RVRR-amc				Pyr-RTKR-amc
		KM	kcat	kcat/KM	kcat/KM variant/wt ratio	KM	kcat	kcat/KM	kcat/KM variant/wt ratio
		μm	s−1	m−1 s−1		μm	s−1	m−1 s−1
Wild-type		28.2 ± 1.3	0.27 ± 0.01	9.2 ± 1.0 × 103	1	4.7 ± 0.5	0.16 ± 0.00	3.4 ± 0.7 × 104	1
187–189	TQM	25.1 ± 2.7	0.03 ± 0.00	1.1 ± 0.2 × 103	0.12 ± 0.03	9.0 ± 1.0	0.05 ± 0.00	6.1 ± 1.1 × 103	0.18 ± 0.07
298–300	REH	20.2 ± 1.2	0.09 ± 0.01	4.5 ± 0.7 × 103	0.49 ± 0.13	5.0 ± 0.4	0.10 ± 0.00	2.1 ± 0.3 × 104	0.62 ± 0.21
345–350	NQNEKQ	23.3 ± 1.9	0.19 ± 0.03	8.1 ± 1.6 × 103	0.88 ± 0.27	5.4 ± 0.3	0.15 ± 0.03	2.7 ± 0.6 × 104	0.73 ± 0.31
357–359	RQK	8.7 ± 0.7	0.03 ± 0.00	2.9 ± 0.3 × 103	0.32 ± 0.07	5.8 ± 0.6	0.08 ± 0.01	1.3 ± 0.2 × 104	0.38 ± 0.14