Table 1 Kinetic parameters of cellulose-active LPMOs measured using different reference reactions.

Reference reaction^a	E and S of interest	[S]	[E]₅₀ (nM)^b	(k_cat/K_m)^app for H₂O₂ (µM⁻¹ s⁻¹)^c
50 nM SmAA10A, 1.0 g L⁻¹ CNW	TrAA9A, Avicel	10 g L⁻¹	432 ± 48	0.17 ± 0.02
		50 g L⁻¹	284 ± 16	0.26 ± 0.01
		100 g L⁻¹	312 ± 18	0.24 ± 0.01
			True k_cat/K_m = 0.27 ± 0.02 µM⁻¹ s^{−1 d}
5 nM HRP, 0.2 mM ABTS	TrAA9A, Avicel	10 g L⁻¹	321 ± 45	0.11 ± 0.02
		50 g L⁻¹	163 ± 23	0.22 ± 0.03
		100 g L⁻¹	156 ± 21	0.23 ± 0.03
1 nM HRP, 0.2 mM ABTS		100 g L⁻¹	35 ± 5	0.21 ± 0.03
			True k_cat/K_m = 0.26 ± 0.01 µM⁻¹ s^{−1 d}
50 nM SmAA10A, 1.0 g L⁻¹ CNW	NcAA9C, Glc₅	0.2 mM	276 ± 6	0.27 ± 0.01
		0.5 mM	165 ± 9	0.46 ± 0.02
		1.0 mM	123 ± 4	0.61 ± 0.02
		2.0 mM	86 ± 3	0.87 ± 0.03
			True k_cat/K_m = 1.19 ± 0.11 µM⁻¹ s^{−1 d}
5 nM HRP, 0.2 mM AmplexRed	NcAA9C, Glc₅	0.1 mM	246 ± 16	0.27 ± 0.02
		0.2 mM	154 ± 11	0.43 ± 0.03
		0.5 mM	87 ± 2	0.77 ± 0.02
		1.0 mM	83 ± 7	0.81 ± 0.06
			True k_cat/K_m = 1.07 ± 0.10 µM⁻¹ s^{−1 d}
10 nM SmAA10A, 1.0 g L⁻¹ CNW	ScAA10C, Avicel	100 g L⁻¹	101 ± 16	0.15 ± 0.02
1 nM HRP, 0.2 mM ABTS	ScAA10C, Avicel	100 g L⁻¹	57 ± 7	0.13 ± 0.02

^aAll reactions were made in 50 mM Bis–Tris buffer pH 6.1 at 25 °C and contained glucose oxidase, 10 mM glucose and 0.1 mM ascorbic acid.
^bHalf-inhibiting concentrations of the enzyme of interest were calculated from the measured rates of the reference reaction in the absence (V_lim) and presence (v_ref) of the enzyme of interest, according to Eq. (5).
^c(k_cat/K_m)^app for the enzyme of interest were calculated from the concentration of the reference enzyme, the [E]₅₀ and the (k_cat/K_m)^app of the reference enzyme for H₂O₂, using Eq. (6). The (k_cat/K_m)^app values of the reference enzymes used in calculations were 1.5 ± 0.7, 7.1 ± 1.2, and 13.4 ± 0.5 µM⁻¹ s⁻¹ for the SmAA10A/CNW, HRP/ABTS, and HRP/AmplexRed reference reactions, respectively. The SD is for the measurement of (k_cat/K_m)^app of the enzyme of interest and does not include the SD of (k_cat/K_m)^app of the reference enzyme.
^dThe true k_cat/K_m values for H₂O₂ were found from nonlinear regression analysis of the dependency of (k_cat/K_m)^app on [S] according to Eq. (7).

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