Table 2.

Activities and kinetic parameters of the wild-type and mutant HsIDH3^a.

Enzyme	Vmax (μmol/min/mg) −activators/+activators	S0.5,ICT (mM) −activators/+activators	Activation effect (fold)b
αβ	2.72 ± 0.14/2.80 ± 0.23	3.65 ± 0.39/3.63 ± 0.62	1.0
αγ	9.62 ± 0.23/16.1 ± 1.1	5.42 ± 0.71/0.26 ± 0.07	20.8
α2βγ	28.6 ± 0.3/30.6 ± 1.0	3.54 ± 0.18/0.43 ± 0.03	8.2
Pseudo allosteric site
M1: αβR99Aαγ	24.9 ± 0.8/27.7 ± 1.5	1.45 ± 0.07/0.34 ± 0.05	4.3
M2: αβY137Aαγ	30.2 ± 1.0/39.4 ± 3.8	1.50 ± 0.09 /0.49 ± 0.05	3.1
M3: αβR274Aαγ	35.9 ± 1.2/38.0 ± 2.4	0.91 ± 0.01/0.23 ± 0.06	4.0
Allosteric site
M4: αβαγR97A	33.2 ± 0.8/34.4 ± 2.1	3.46 ± 0.29/1.20 ± 0.09	2.9
M5: αβαγY135A	27.8 ± 1.1/33.2 ± 4.2	2.28 ± 0.23/2.16 ± 0.12	1.1
M6: αβαγR272A	27.5 ± 0.8/34.1 ± 3.3	2.40 ± 0.20/2.80 ± 0.30	0.9
Heterodimer interface
M7: αβK153Aαγ	9.21 ± 0.43/18.4 ± 2.0	3.13 ± 0.49/1.80 ± 0.13	1.7
M8: αβαγK151A	5.24 ± 0.63/9.19 ± 2.12	6.96 ± 0.50/10.2 ± 3.1	0.7
M9: αK142Aβαγ	6.53 ± 0.44/6.84 ± 0.45	2.50 ± 0.35/0.76 ± 0.03	3.3
M10: αβαK142Aγ	3.33 ± 0.11/4.20 ± 0.61	1.55 ± 0.10/0.84 ± 0.04	1.8
M11: αK142AβαK142Aγ	1.92 ±± 0.40/1.48 ± 0.22	11.42 ± 2.01/8.50 ± 2.50	1.3
Heterodimer–heterodimer interface
M12: αβE150Aαγ	15.2 ± 0.7/25.1 ± 1.0	1.34 ± 0.05/0.24 ± 0.07	5.6
M13: αβαγE148A	13.4 ± 0.4/14.0 ± 1.7	1.29 ± 0.05/0.25 ± 0.12	5.2
M14: αQ139Aβαγ	36.1 ± 1.9/43.4 ± 2.3	0.52 ± 0.04/0.35 ± 0.04	1.5
M15: αβαQ139Aγ	33.0 ± 1.2/36.4 ± 0.6	0.40 ± 0.05/0.23 ± 0.03	1.7
M16: αQ139AβαQ139Aγ	39.7 ± 0.9/47.4 ± 1.6	0.24 ± 0.03/0.18 ± 0.03	1.3
Deletion of the N-terminal of the γ subunit (ΔN)
M17: αγΔN	9.51 ± 0.16/18.2 ± 0.47	5.73 ± 0.40/0.46 ± 0.04	12.5
M18: αβαγΔN	5.50 ± 0.21/13.0 ± 0.56	3.85 ± 0.43/2.02 ± 0.09	1.9
Substitution of the C-terminal of the β subunit (βmut)
α2βmutγ	27.7 ± 0.6/34.0 ± 1.5	2.82 ± 0.21/0.40 ± 0.08	7.1

^aThe enzymatic activity and kinetic data were measured at standard conditions with varied concentrations of ICT in the absence or presence of the activators (CIT and ADP).

^bActivation effect = S_0.5,ICT (no activators)/S_0.5,ICT (+activators).