TABLE 2.

Substrate specificity of OPB

To test the predictive power of the PS-SCL, fluorogenic substrates were obtained that were predicted to be either good or poor matches by the PS-SCL results. Substrates were grouped and listed by decreasing initial cleavage velocities. Percentage activity is normalized to the initial velocity observed using the Z-PR-AMC substrate. All assays were performed at pH 8.0 using 20 μm substrate (except for those used to calculate K_cat and K_m values). The PS-SCL predicted that the ideal P2 amino acid would be Gly. Therefore, Z-GGR-AMC and Z-GPR-AMC, which differ only in the P2 position, were compared as substrates for OPB, and the kinetic parameters were calculated. These assays were performed at substrate concentrations ranging from 80 to 0.2 μm.

N-terminal group	P5	P4	P3	P2	P1	Relative activity	N-terminal group	P5	P4	P3	P2	P1	Relative activity
						%							%
Z			G	G	R	197	H					A	0
Boc			L	G	R	108	Ac		D	E	V	D	0
Z			G	P	R	104	Z			V	A	D	0
Z				P	R	100	Ac		W	E	H	D	0
Z			V	V	R	92	Z			L	L	E	0
Glut				G	R	91	Suc			A	A	F	0
Z				R	R	89	Suc		P	S	P	F	0
Z				L	R	78	H				G	F	0
Boc			L	R	R	69	H					L	0
Ac				F	R	62	Z			G	G	L	0
Ac	K	Q	K	L	R	51	Z			A	E	N	0
Z				F	R	50	Ac			A	S	N	0
Z		A	A	R	R	40	Ac			A	V	N	0
Boc			E	K	K	32	Suc	G	P	L	G	P	0
Boc		R	V	R	R	31	Suc				L	Y	0
Ac	F	R	S	L	K	23	Suc		L	L	V	Y	0
Boc			V	R	K	21	TFA					Y	0
H					R	6
H					K	1

Kinetic parameter	Z-GPR-AMC	Z-GGR-AMC
Km (μm)	7.76	3.08
Kcat (s−1)	4.25e4	6.83e4
Kcat/Km (s−1 μm−1)	0.55e4	2.22e4