Table 3. Steady-state kinetic parameters for peptidase activity with λN 89-98 peptide variants.

From the FRETN 89-98 model substrate, additional peptides were synthesized to identify the residues important for activity. Steady-state kinetic parameters for peptidase activity were determined using the fluorogenic peptidase assay as described in materials and methods. Class I peptides represent the alanine scan, in which each residue, in turn, was replaced by an alanine while other residues remained the same. Of the Class I peptides, alanine substitutions at I91 (P3), C93 (P1), and R96 (P3’) showed a large decrease in the k_cat/K_m values, identifying these residues as important for maximal peptidase activity. Class II peptides contain substitutions of the three residues that were identified as integral to peptidase activity. Maximum activity occurred in C2-005, where the only non-alanine residues were at I91, C93 and R96. In the peptide C2-003, with alanine substitution at all three residues, peptidase activity was practically abolished. Class III peptides contained substituted at the cleavage site, indicating the importance of the C93 (P1).

Class I peptides	kcat (s-1)	Km (μM)	kcat/Km (×104 M-1s-1)
FRETN 89-98 R89A	8.5 ±0.4	165 ± 38	5.1
FRETN 89-98 G90A	7.2 ± 0.6	104±36	6.9
FRETN 89-98 I91A	2.1 ±0.3	168±69	1.2
FRETN 89-98 T92A	6.2 ±0.5	121±41	5.1
FRETN 89-98 C93A	3.3±0.3	146±47	2.2
FRETN 89-98 S94A	6.7±0.8	80±40	8.4
FRETN 89-98 G95A	8.7±0.6	123±38	7.0
FRETN 89-98 R96A	2.5±0.2	117±39	2.1
FRETN 89-98 Q97A	5.6±0.8	99±51	5.6
Class II peptides
FRETN 89-98 I91R, R96I (C2-001)	0.33±0.03	156±58	0.21
FRETN 89-98 I91A, R96A (C2-002)	0.24±0.03	199±83	0.12
FRETN 89-98 I91A, C93A, R96A (C2-003)	0.16±0.05	325±200	0.04
FRETN 89-98 All Ala except I91, R96 (C2-004)	6.7±0.1	133±75	5.0
FRETN 89-98 All Ala except I91, C93, R96 (C2-005)	4.7±0.2	56±15	8.4
Class III peptides
FRETN 89-98 C93G	0.031±0.01	206±98	0.01
FRETN 89-98 C93S	0.051±0.01	208±92	0.02
FRETN 89-98 S94G	3.67±0.57	196±92	1.9