TABLE 2.

Kinetic parameters for the E78Q, E122Q, and E78Q/E122Q

Kinetic parameters were determined in both reaction directions for E78Q and E122Q. For E78Q/E122Q mutant enzyme it was determined only in the direction of saccharopine formation. The reactions were monitored at 25 °C and at pH 7.2.

Kinetic parameters at pH 7.2	SDH-WT	E78Q	E122Q	E78Q/E122Q
Forward reaction
V1/Et (s−1)	1.1 ± 0.1	3.95 ± 0.01	0.76 ± 0.04	NDa
Fold change		+3.6	−1.45
V1/KNADEt (m−1s−1)	(1.2 ± 0.1) × 103	(3.62 ± 0.58) × 103	(2.31 ± 0.3) × 102	ND
Fold change		+ 3.1	−5.2
V1/KSacEt (m−1s−1)	(1.6 ± 0.3) × 102	(2.1 ± 0.4) × 103	(5.5 ± 0.3) × 101	ND
Fold change		+13	−3	ND
KSac (mm)	6.7 ± 1.4	2.0 ± 0.4	14.0 ± 0.8	ND
Fold change		−3.4	+2.1
KNAD (mm)	0.9 ± 0.1	1.1 ± 0.2	3.3 ± 0.4	ND
Fold change		+1.2	+3.7
KiNAD (mm)	1.1 ± 0.3	0.5 ± 0.3	1.9 ± 0.3	ND
Fold change		−2.0	+1.7	ND
Reverse reaction
V2/Et (s−1)	20.0 ± 1.0	11.2 ± 0.4	4.3 ± 0.1	24.7 ± 2.6
Fold change		−1.2	−4.6	+1.2
V2/KNADHEt (m−1s−1)	(1.6 ± 0.2) × 106	(8.0 ± 0.3) × 105	(1.9 ± 0.2) × 105	(2.0 ± 0.2) × 105
Fold change		−2	−8.4	−8
V2/KLysEt (m−1s−1)	(2.5 ± 0.4) × 104	(2.8 ± 0.2) × 103	(4.100 ± 0.001) × 102	(9.1 ± 0.9) × 102
Fold change		−8.8	−60.7	−27.4
V2/Kα-KgEt (m−1s−1)	(2.8 ± 0.7) × 105	(4.9 ± 0.3) × 104	(1.50 ± 0.07) × 104	(1.3 ± 0.1) × 104
Fold change		−5.7	−18	−22
KNADH (mm)	0.019 ± 0.002	0.014 ± 0.004	0.025 ± 0.002	0.12 ± 0.01
Fold change		−1.4	+1.3	+6.3
KLys (mm)	1.1 ± 0.2	4.0 ± 0.4	11.0 ± 0.6	27.1 ± 1.3
Fold change		+3.6	+10	+24.5
Kα-Kg (mm)	0.11 ± 0.03	0.23 ± 0.02	0.30 ± 0.01	2.0 ± 0.5
Fold change		+2.1	+2.7	+17.8

^a ND, not determined.