Table 2.

Steady‐state kinetic parameters of the wild‐type and variant enzymes.^a

Protein variant	Kinetic parameters for pNPC2			Kinetic parameters for MF			Substrate discrimination
	Km (mM)	kcat (s−1)	kcat/Km (s−1 M−1)	Km (mM)	kcat (s−1)	kcat/Km (s−1 M−1)	(kcat/Km)pNPC2/(kcat/Km)MF
3A6	0.18 ± 0.09	137.4 ± 1.5	763	2.34 ± 0.37	104.3 ± 0.5	45	17
K281N	0.31 ± 0.03	129.2 ± 0.9	417	2.02 ± 0.14	337.0 ± 1.1	167	2.5
K281T	0.27 ± 0.03	155.3 ± 1.6	575	2.07 ± 0.26	296.3 ± 0.9	143	4.0
K281S	0.29 ± 0.03	171.8 ± 1.3	592	2.40 ± 0.60	277.5 ± 0.7	116	5.1
K281I	0.66 ± 0.03	715.2 ± 5.0	1084	0.16 ± 0.05	242.2 ± 0.7	1514	0.7
D282E	0.13 ± 0.03	147.0 ± 0.7	1131	0.93 ± 0.16	696.8 ± 0.4	749	1.5
D282L	0.11 ± 0.04	278.7 ± 2.6	2537	1.89 ± 0.10	69.4 ± 0.8	37	68.6
N316L	0.22 ± 0.02	109.9 ± 0.6	500	2.16 ± 0.48	235.8 ± 1.0	109	4.6
N316STOP	0.18 ± 0.04	161.4 ± 2.2	1494	8.79 ± 0.89	3.20 ± 0.05	0.4	3735.0
K317N	0.31 ± 0.04	88.0 ± 0.6	284	2.38 ± 0.14	517.4 ± 0.3	217	1.3
K317G	0.24 ± 0.02	151.2 ± 1.2	630	1.92 ± 0.58	475.9 ± 0.4	248	2.5
K317L	0.10 ± 0.01	138.8 ± 1.2	578	2.77 ± 0.49	429.8 ± 0.5	155	3.7
K317D	0.24 ± 0.03	136.0 ± 1.3	567	0.93 ± 0.35	423.5 ± 0.8	455	1.2
K317H	0.39 ± 0.12	129.3 ± 1.0	331	22.51 ± 3.50	3.6 × 10−3	1.6 × 10−4	2 × 106
3A6I	3.48 ± 0.21	164.1 ± 1.9	47	32.20 ± 4.41	5.8 × 10−3	1.8 × 10−4	26111
K281I/D282E	0.16 ± 0.07	493.7 ± 3.4	3086	0.72 ± 0.08	3494.1 ± 2.9	4853	0.6
D282L/N316STOP	0.25 ± 0.06	961.5 ± 5.4	3844	5.81 ± 0.60	226.6 ± 1.8	39	98.6
D282L/K317H	0.12 ± 0.03	270.4 ± 3.1	2253	8.60 ± 1.50	17.2 ± 2.7	2	1126.5
N316STOP/K317H	0.07 ± 0.01	181.0 ± 4.7	2587	17.50 ± 1.90	2.3 × 10−3	1.6 × 10−4	1.6 × 106
D282L/N316STOP/K317H	0.12 ± 0.03	779.2 ± 3.8	6493	16.50 ± 2.51	93.0 ± 1.7	6	1082.2
K281I/D282E/K317D	0.48 ± 0.05	1611.8 ± 8.5	3358	0.39 ± 0.07	3171.8 ± 8.3	8133	0.4
A8P4 (H26/A85P/T86P)	2.29 ± 0.62	9826.7 ± 9.0	4279	2.04 ± 0.10	7.3 ± 0.2	3.6	1188

^a.Reaction conditions: [E]_o = 0–12 nM, [substrate] ranging from 0 to 50 mM, 100 mM Tris‐sulfate, pH 8.5, T = 40°C.