Table 1.

Apparent steady‐state kinetic constants of PDH from A. meleagris and its variants for the electron donor GLC and the electron acceptor Fc⁺. GLC was probed with the standard Fc⁺ assay (50 mm sodium phosphate buffer, pH 7.5, 30 °C, fixed value of 0.2 mm Fc⁺ as electron acceptor). Fc⁺ was tested in 100 mm borate buffer, pH 8.5, 30 °C. The fixed GLC concentration was adjusted according to the K _M value of the corresponding variant: 25 mm (nsAmPDH, recAmPDH, AmPDH), 50 mm (H103A, Q392A, V511W), 250 mm (Y510A) or 500 mm (H556A, H556N, V511F). ND, not determined

Variant	GLC			Fc+
	K M (mm)	k cat (s−1)	k cat/K M (mm −1·s−1)	K M (mm)	k cat (s−1)	k cat/K M (mm −1·s−1)
nsAmPDHc	0.82 ± 0.03	45.9 ± 0.3	57.5	0.13 ± 0.03	104 ± 8	802
recAmPDHa	0.69 ± 0.09	37.8 ± 1.1	54.8	0.16 ± 0.04	130 ± 11	812
AmPDHb	0.82 ± 0.01	42.2 ± 0.2	51.5	0.17 ± 0.02	135 ± 7	776
H556N	97.5 ± 4.1	6.40 ± 0.08	0.07	0.47 ± 0.04	22.5 ± 1.3	47.8
H556A	90.0 ± 3.5	12.8 ± 0.2	0.14	0.17 ± 0.01	20.4 ± 0.5	124
H512A	6.19 ± 1.24	0.0010 ± 0.0001	0.0002	ND	ND	ND
V511W	3.73 ± 0.17	18.6 ± 0.2	5.00	0.11 ± 0.01	38.0 ± 1.4	352
V511F	106 ± 5	19.9 ± 0.3	0.19	0.39 ± 0.03	26.4 ± 1.4	66.8
Y510A	60.7 ± 2.0	16.0 ± 0.2	0.26	0.49 ± 0.03	28.8 ± 1.3	59.2
Q392A	6.15 ± 0.21	24.5 ± 0.2	3.98	0.30 ± 0.02	111 ± 4	372
H103A	2.73 ± 0.11	15.6 ± 0.1	5.70	0.16 ± 0.01	34.5 ± 1.3	222

^a AmPDH produced in the natural source A. meleagris 2.

^b AmPDH recombinantly produced in P. pastoris 3.

^c AmPDH with a His₆‐tag, recombinantly produced in P. pastoris (present study).