Table 1.

Kinetic parameters—K _m (µM), k _cat (s⁻¹), and k _cat /K _m (s⁻¹ mM⁻¹)—for the oxidation of Mn²⁺, phenolic (DMP) and nonphenolic (VA) aromatics, and dyes (means and 95% confidence limits) by the resurrected enzymes and extant LiPH8 (a comparison of all catalytic efficiencies is shown in Fig. 4)

	CaPo	CaCD	AVP	ALiP	LiPH8
Metal ion
Mn2+
K m	700 ± 48	275 ± 41	62 ± 10	–a	–
k cat	185 ± 3	170 ± 6	106 ± 4	–	–
k cat /K m	260 ± 15	617 ± 80	1710 ± 240	–	–
Aromatics
DMPb (low efficiency)
K m	32,900 ± 2700	66,800 ± 3800	32,500 ± 12,100	–	–
k cat	221 ± 9	109 ± 4	31 ± 5	–	–
k cat /K m	6.7 ± 0.3	1.6 ± 0.03	1.0 ± 0.2	–	–
DMP (high efficiency)
K m	–	–	5.3 ± 1.1	34.0 ± 5.4	4.0 ± 0.07
k cat	–	–	4.5 ± 0.1	18.3 ± 0.7	6.9 ± 0.5
k cat /K m	–	–	837 ± 162	537 ± 55	600 ± 36
VA
K m	–	–	299 ± 104	773 ± 155	79.3 ± 18
k cat	–	–	7.1 ± 0.5	21.3 ± 1.0	16.2 ± 0.8
k cat /K m	–	–	24 ± 7	28 ± 5	205 ± 4
Dyes
ABTSb (low efficiency)
K m	3170 ± 270	1280 ± 350	2150 ± 420	–	–
k cat	539 ± 24	103 ± 10	25 ± 2	–	–
k cat /K m	170 ± 8	80 ± 15	12 ± 1	–	–
ABTS (high efficiency)
K m	–	–	5.4 ± 0.7	13.7 ± 3.5	21.3 ± 2.3
k cat	–	–	2.1 ± 0.1	12.5 ± 0.7	6.5 ± 0.2
k cat /K m	–	–	400 ± 44	911 ± 212	300 ± 25
RB5
K m	–	–	4.8 ± 0.8	12.6 ± 3.6	–
k cat	–	–	2.4 ± 0.2	5.4 ± 0.8	–
k cat /K m	–	–	504 ± 50	428 ± 68	–
Direction of evolution

The alternative CaPo ancestors oxidized DMP (low efficiency) with K _m 455,000 ± 84,000 (CaPo-bis) and 298,000 ± 66,000 μM (CaPo-tris), k _cat 129 ± 22 (CaPo-bis) and 108 ± 21 s⁻¹(CaPo-tris), and k _cat /K _m 0.3 ± 0.05 (CaPo-bis) and 0.4 ± 0.01 s⁻¹mM⁻¹ (CaPo-tris); ABTS (low efficiency) with K _m, 1392 ± 345 (CaPo-bis) and 807 ± 104 μM (CaPo-tris), k _cat, 241 ± 23 (CaPo-bis) and 128 ± 6 s⁻¹ (CaPo-tris), and k _cat /K _m, 173 ± 28 (CaPo-bis) and 160 ± 15 s⁻¹  mM⁻¹ (CaPo-tris), while Mn²⁺ was only oxidized by CaPo-tris with K _m 5010 ± 274 µM, k _cat 47 ± 1 s⁻¹, and k _cat /K _m 9.3 ± 0.4 s⁻¹ mM⁻¹

Reactions at 25 °C in 0.1 M tartrate at optimal pH 5.0 (CaPo, and its alternative ancestors, and CaCD) or 5.5 (AVP) for Mn²⁺, pH 3.5 (AVP) or 3.0 (ALiP and LiPH8) for VA, pH 2 (CaPo) or 3.0 (others) for DMP, pH 3.0 (CaPo) and pH 3.5 (others) for ABTS, and pH 3 for RB5; and saturating H₂O₂ concentrations of 0.4 mM for CaPo, CaCD, and AVP, 0.2 mM for ALiP, and 0.1 mM for LiPH8

^a–Absence of activity

^bBiphasic kinetics for DMP and ABTS oxidation by AVP enabled calculation of two sets of constants assigned to two catalytic sites, as reported for extant VP [40], comparable to those present in CaPo/CaCD and ALiP/LiPH8, respectively