Table 2.

Steady-state kinetic parameters of cancer-associated IDH1/2 variants for 2OG and selected 2OG derivatives

Entry	2OG derivative	a,bParameter	R132H IDH1	R172K IDH2	R140Q IDH2
A		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	0.72 ± 0.04	0.72 ± 0.04	0.49 ± 0.04
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	0.85 ± 0.19	0.35 ± 0.04	5.1 ± 1.2
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	0.85 ± 0.22	2.1 ± 0.3	0.10 ± 0.03
B		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	6.9 ± 0.4	4.8 ± 0.2	5.4 ± 0.3
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	3.7 ± 0.1	4.2 ± 0.4	2.0 ± 0.8
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	1.9 ± 0.1	1.2 ± 0.1	2.8 ± 1.1
C		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	8.3 ± 0.6	3.9 ± 0.2	8.6 ± 0.4
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	8.5 ± 1.2	15 ± 1	5.0 ± 0.5
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	0.99 ± 0.16	0.26 ± 0.03	1.7 ± 0.2
D		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	12 ± 1	2.7 ± 0.2	8.9 ± 0.8
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	13 ± 1	11 ± 5	9.4 ± 2.3
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	0.98 ± 0.06	0.24 ± 0.11	0.95 ± 0.25
E		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	9.2 ± 0.4	7.8 ± 1.8	9.7 ± 0.4
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	5.7 ± 3.3	16 ± 5	3.8 ± 0.8
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	1.6 ± 1.0	0.50 ± 0.19	2.6 ± 0.6
F		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	5.7 ± 0.6	1.9 ± 0.2	7.2 ± 0.6
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	7.2 ± 0.5	25 ± 1	7.5 ± 3.6
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	0.80 ± 0.10	0.079 ± 0.007	0.97 ± 0.48
G		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	5.3 ± 0.4	3.9 ± 0.3	6.0 ± 0.5
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	12 ± 1	25 ± 5	38 ± 3
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	0.44 ± 0.06	0.16 ± 0.04	0.16 ± 0.02
cH		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	3.4 ± 0.3	3.7 ± 0.2	8.0 ± 0.6
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	15 ± 2	16 ± 1	23 ± 7
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	0.23 ± 0.04	0.23 ± 0.02	0.35 ± 0.11
I		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [s-1]	5.7 ± 0.4	3.0 ± 0.2	5.8 ± 0.6
		${\mathit{K}}_{\mathbf{m}}^{\mathbf{a}\mathbf{p}\mathbf{p}}$ [mM]	12 ± 5	16 ± 2	14 ± 2
		${\mathit{k}}_{\mathbf{c}\mathbf{a}\mathbf{t}}$/${\mathit{K}}_{\mathbf{m}}$ [mM-1·s-1]	0.47 ± 0.21	0.19 ± 0.03	0.43 ± 0.08

^a Mean of two independent runs, each composed of technical duplicates (n = 2; mean ± SD).

^b IDH1/2 variant assays monitoring NADPH turnover were performed as described in the Experimental procedures section using 0.03 μM IDH1/2 variant and 50 μM NADPH in reaction buffer (100 mM Tris, pH 8.0, 0.005%_v/v tween-20, 0.1 mg/mL BSA, 0.2 mM DTT, 20 mM MgCl₂, 150 mM NaCl).

^c mixture of racemic diastereomers, dr (cis:trans) = 2.5:1.