Table 2.

Steady-state kinetic parameters—K_M (µM), k_cat (s⁻¹), k_cat/K_M (s⁻¹·mM⁻¹), and k_i (µM)—for H₂O₂, ABTS, DMP, VA, and RB5 reactions of ApeLiP and its W166A variant.

			ABTS a		DMP
		H2O2	Low Efficiency	High Efficiency	pH 3	pH8	VA	RB5
ApeLiP	K M	33 ± 4.4	693 ± 63	8.1 ± 0.6 b	37 ± 5.6	1430 ± 440	(29.6 ± 4.6) × 103	14 ± 1.4 b
	k cat	100 ± 4.8	179 ± 5	42 ± 1.8	68.6 ± 2.8	5.7 ± 0.7	4.3 ± 0.3	21 ± 1.5
	kcat/KM	3070 ± 440	258 ± 25	5130 ± 460	1910 ± 310	4.0 ± 1.3	0.15 ± 0.03	1540 ± 115
	k i	(2.1 ± 0.5) × 103	-	-	(11 ± 2.2) × 103	nd d	(406 ± 92) × 103	-
W166A	K M	136 ± 12	1150 ± 190 c	-	-	49 ± 8.2	-	-
	k cat	126 ± 4.7	117 ± 10 c	-	-	6.1 ± 0.27	-	-
	kcat/KM	929 ± 87	102 ± 19 c	-	-	125 ± 22	-	-
	k i	(3.3 ± 0.3) × 103	nd c	-	-	nd d	-	-

Reactions in 100 mM sodium tartrate or Tris-HCl buffer at 25 °C under H₂O₂ saturation (0.4 mM for ApeLiP and 1 mM for W166A). H₂O₂ reduction (estimated with 5 mM ABTS as reducing substrate) and ABTS and RB5 oxidation were measured at pH 3.0, DMP oxidation at pH 3.0 and 8.0, and VA oxidation at pH 4.0. Unless otherwise stated, Michaelis–Menten or inhibition equations were used. ^a Biphasic kinetics yielding two sets of constants. ^b Data from high-efficiency oxidation of ABTS and RB5 adjusted to Hill equation (n = 1.62 ± 0.12 and 2.11 ± 0.30, respectively; K_0.5 considered equivalent to K_M). ^c,d Substrate/product inhibition with parameters estimated from Hill (n = 1.62 ± 0.28) and Michaelis–Menten equations, respectively, as data did not fit to the inhibition equation. nd, not determined. -, no activity. Means and standard deviations are shown.