TABLE 2.

Structural and steady-state kinetic data for native and mutant LF substrates selected from a phage library

Kinetic parameters for substrates were obtained using protein FRET. Peptides were engineered as linkers between fluorescent proteins. Cleavage of the linker separated the FRET pair members PSCFP2 and phiYFP, resulting in loss of FRET signal and enabling fluorometric analysis of the reaction progression. Amino acid sequences of peptide substrates are numbered P8 to P4′ from the N to C terminus. S indicates substrates were isolated from the phage library. LF15P is the protein FRET substrate constructed based on the LF15 peptide (7). MKK6P was prepared using the LF cleavage site in Mkk6 kinase (18). Amino acid positions subjected to site-directed mutagenesis are underlined. Kinetic data were collected in triplicate and fitted using Sigma Plot enzyme Kinetics software.

Mut no.	Description of substrate	Peptide substrate composition												Kinetic parameters
		P8	P7	P6	P5	P4	P3	P2	P1	P1′	P2′	P3′	P4′	Km·10−6	kcat·10−4	kcat/Km
														m	s−1	m−1 s−1
	LF15P	K	R	R	K	K	V	Y	P	Y	P	M	E	5.4 ± 0.5	(5 ± 0.3)·103	92593
	MKK6P	K	K	R	N	P	G	L	K	I	P	K	E	29.0 ± 3.7	6.1 ± 1.2	21
	S82	R	I	I	R	R	V	N	S	S	L	P	L	15.7 ± 2.3	0.9 ± 0.2	6
	S40	R	D	F	R	R	I	I	A	E	R	Y	L	6.6 ± 1.3	1.9 ± 0.3	28
	S20	R	D	I	R	R	I	T	L	F	S	L	H	3.4 ± 0.7	5.0 ± 0.5	147
1	S20 Lp1P	R	D	I	R	R	I	T	P	F	S	L	H	17.1 ± 4.2	2.1 ± 0.4	12
2	S20 Fp1′Y	R	D	I	R	R	I	T	L	Y	S	L	H	2.1 ± 0.7	0.8 ± 0.2	38
3	S20 Ip3V/Tp2Y/Lp1P	R	D	I	R	R	V	Y	P	F	S	L	H	4.2 ± 1.2	0.9 ± 0.1	22.7
4	S20 Dp7K/Ip6K	R	K	K	R	R	I	T	L	F	S	L	H	86.8 ± 10.2	3.93 ± 0.8	4.53
5	S20 Dp7K/Ip6K/Ip3V/Tp2Y	R	K	K	R	R	V	Y	L	F	S	L	H	1.0 ± 0.1	9.8 ± 0.3	946
6	S20 Dp7K/Ip3V/Tp2Y/Lp1P/Sp2′P	R	K	I	R	R	V	Y	P	F	P	L	H	5.9 ± 2.0	116.0 ± 33.7	1960