Table 3. TaTT variants and their initial activities in the overall reactions between R-PEA and pyruvate, R-PEA and α-ketoglutarate, L-leucine and α-ketoglutarate.

TaTT	Mutations	Initial activity, U/mg			Tm, °C
		R-PEA + pyruvatea	R-PEA + α-ketoglutaratea	L-Leu + α-ketoglutarateb
WT	No	0.124 ± 0.005	0.147 ± 0.003	40 ± 5	88.0 ±0.6
		0.004 ± 0.001c	0.002 ± 0.001c	23 ± 6c
mP1	R43S	0.095 ± 0.004	0.14 ± 0.01	0.79 ± 0.02	85.8 ± 0.4
mP2	G41V + Y101F	0.004 ± 0.001	0.16 ± 0.01	n.d.	84.2 ± 0.3
mP3	R43S+ G41V + Y101F	0.004 ± 0.001	0.018 ± 0.006	n.d.	86.2 ± 0.2
		0.008 ± 0.001c	0.008 ± 0.001c
mO1	S115R	0.112 ± 0.005	0.064 ± 0.003	1.8 ± 0.4	n.m.
mO2	Y166W + F39Y	n.d.	n.d.	n.d.	83.9 ±0.2
mP2O1	G41V + Y101F + S115R	0.003 ± 0.001	0.004 ± 0.001	n.d.	n.m.
mP3O1	R43S + G41V + Y101F + S115R	0.003 ± 0.001	0.024 ± 0.002	n.d.	82.9 ± 0.4
		0.012 ± 0.008c	0.066 ± 0.003c
mP3O3	R43S + G41V + Y101F + S115R + A108	0.007 ± 0.002c	n.d.	n.m.	n.m.
mP3O4	R43S + G41V + Y101F + S115R + Y166W + F39Y	n.d.	n.d.	n.d.	n.m.
mP3O5	R43S + G41V + Y101F + W32H	n.m.	0.008 ± 0.001	n.d.	n.m.
mP3O6	R43S + G41V + Y101F + F39Y	n.d.	n.d.	n.d.	n.m.
mP3O7	R43S + G41V + Y101F + F39Y + W32H	n.m.	0.003 ± 0.001	n.d.	n.m.

Variants containing changes in P-pocket are named mP1-mP3; variants containing changes in O-pocket are named mO1-mO2; variants including changes in both pockets are named mPxOx accordingly. Apparent melting temperature is shown as well.

^aInitial activity measured at pH 9.0 using the acetophenone assay

^bInitial activity measured at pH 8.0 using the GDH assay

^cThe reaction was carried out at pH 7.0 (50 mM Tris-HCl, 50°C)

n.m. = not measured

n.d. = not detected. The detection limit of the acetophenone assay is 0.0001 U/mg. The detection limit of the GluDH assay is 0.08 U/mg.