Table 6.

Stopped‐flow spectroscopy for the reductive half‐reaction of AmPDH with GLC, GAL, ARA and MGP and the oxidative half‐reaction with BQ. For variant H556A, only GLC and BQ were used. Experiments were performed in 50 mm potassium phosphate buffer (pH 7.0) and 4 °C. Stopped‐flow traces were simulated with berkeley madonna, version 8.3.14, according to Kinetic Model 1_red or Kinetic Model 1_ox. Employed enzyme and substrate concentrations, monitored wavelengths and experimental and simulated traces are provided in Figs 6 and 7. Additional details on GC‐MS measurements to determine the GLC oxidation sites for H556A are provided in Table 4. y‐axis intercepts for AmPDH were 7.61 (GLC), 9.11 (GAL), 9.06 (ARA), 15.2 (MGP) and 0.92 (BQ). y‐axis intercepts for H556A were zero for GLC and BQ

Variant	Substrate	Oxidation site	Reference for oxidation site	Apparent bimolecular rate constant k app (m −1·s−1)
				Experiment	Simulation
AmPDH	GLC	C2/3a	11, 16	5.4 ± 0.11 × 104	6.9 × 104
	GAL	C2	16	4.4 ± 0.15 × 104	5.2 × 104
	ARA			6.8 ± 0.10 × 104	7.8 × 104
	MGP	C3	12	4.8 ± 0.05 × 104	6.5 × 104
	BQ	–	–	6.6 ± 0.17 × 103	8.0 × 103
H556A	GLC	C2/3a	Present study	7.3 ± 0.24 × 101	8.0 × 101
	BQ	–	–	4.1 ± 0.02 × 104	3.6 × 104

^aPredominantly oxidized at C2 and only very slowly at C3 (Table 4).