TABLE 2.

Kinetic parameters of LepB and LepA

Substrate	LepB			LepA
	Km (μM)	kcata (s−1)	kcat/Km (μM−1 s−1)	Km (μM)	kcata (s−1)	kcat/Km (μM−1 s−1)
Boc-Val-Leu- Lys-MCAb	11	168	15	1.3	123	95
Bz-Lys-pNAc	67	3.0	0.045	65	5.7	0.088
Ac-Lys-pNAc	42	14	0.33	18	4.5	0.25
Lys-pNAc	100	0.21	0.0021	77	0.18	0.0023

^aParameters were determined by Lineweaver-Burk plots, and the molecular masses of 28,916.2 and 27,735.9 Da calculated from the deduced amino acid sequences were used for the k_cat values of LepB and LepA, respectively.

^bReaction mixtures containing 0.5 to 30 μM Boc-Val-Leu-Lys-MCA and 20 mM Tris-HCl (pH 9.0) were incubated at 30°C for 5 min, and the reaction was initiated by the addition of LepB or LepA to a final concentration of 1 pM in a total volume of 3 ml. Increased fluorescence of liberated 7-amino-4-methylcoumarin was monitored at 440 nm upon excitation at 380 nm for 4 min.

^cReaction mixtures containing 40 to 250 μM Bz-Lys-pNA, 20 to 200 μM Ac-Lys-pNA, or 35 to 250 μM Lys-pNA and 180 mM Tris-HCl (pH 9.2) were incubated at 30°C for 5 min, and the reaction was initiated by the addition of LepB or LepA to a final concentration of 16.6 nM for Bz-Lys-pNA, 4.1 nM for Ac-Lys-pNA, and 170 nM for Lys-pNA in a total volume of 0.75 ml. After 10 min of incubation, the reaction was terminated by adding 0.25 ml of 45% (vol/vol) acetic acid, and the absorbance at 405 nm was measured.