Table 1 Effects of magnesium and manganese ions on kinetic parameters for the oxidative and reductive reactions of wt and R132H IDH1, respectively.

Oxidative reaction (isocitrate to 2OG) with Mg²⁺				Reductive reaction (2OG to isocitrate by wt, to 2HG by R132H) with Mg²⁺
		wt	R132H			wt	R132H
Isocitrate	K_M (µM)	38 ± 6	257 ± 50	2OG	K_M (µM)	(1.1 ± 0.3) × 10³	652 ± 116
	V_max (nM s⁻¹)	216 ± 8	36 ± 2		V_max (nM s⁻¹)	200 ± 16	236 ± 10
	k_cat/K_M (s⁻¹ µM⁻¹)	1.3 ± 0.3	(3.9 ± 1.1) × 10⁻⁴		k_cat/K_M (s⁻¹ µM⁻¹)	(1.9 ± 0.6) × 10⁻³	(9.4 ± 2.7) × 10⁻⁴
NADP⁺	K_M (µM)	27 ± 2	10 ± 1	NADPH	K_M (µM)	115 ± 19	15 ± 2
	V_max (nM s⁻¹)	246 ± 4	40 ± 1		V_max (nM s⁻¹)	221 ± 15	276 ± 10
	k_cat/K_M (s⁻¹ µM⁻¹)	1.9 ± 0.3	(1.0 ± 0.2) × 10⁻²		k_cat/K_M (s⁻¹ µM⁻¹)	(1.8 ± 0.4) × 10⁻²	(4.1 ± 1.0) × 10⁻²
MgCl₂	K_M (µM)	36 ± 4	171 ± 23	MgCl₂	K_M (µM)	^a	(4.4 ± 0.7) × 10³
	V_max (nM s⁻¹)	214 ± 5	45 ± 2		V_max (nM s⁻¹)		224 ± 10
	k_cat/K_M (s⁻¹ µM⁻¹)	1.4 ± 0.3	(5.8 ± 1.4) × 10⁻⁴		k_cat/K_M (s⁻¹ µM⁻¹)		(1.4 ± 0.4) × 10⁻⁴
	k_cat (s⁻¹)	50 ± 4	0.10 ± 0.01		k_cat (s⁻¹)	2.1 ± 0.1	0.61 ± 0.07

Oxidative reaction (isocitrate to 2OG) with Mn²⁺				Reductive reaction (2OG to isocitrate by wt, to 2HG by R132H) with Mn²⁺
		wt	R132H			wt	R132H
Isocitrate	K_M (µM)	35 ± 4	219 ± 35	2OG	K_M (µM)	^b	175 ± 26
	V_max (nM s⁻¹)	313 ± 8	355 ± 22		V_max (nM s⁻¹)		390 ± 16
	k_cat/K_M (s⁻¹ µM⁻¹)	2.3 ± 0.6	(3.6 ± 0.9) × 10⁻³		k_cat/K_M (s⁻¹ µM⁻¹)		(5.9 ± 1.2) × 10⁻³
NADP⁺	K_M (µM)	27 ± 3	80 ± 8	NADPH	K_M (µM)		32 ± 5
	V_max (nM s⁻¹)	358 ± 7	302 ± 9		V_max (nM s⁻¹)		433 ± 18
	k_cat/K_M (s⁻¹ µM⁻¹)	3.0 ± 0.7	(9.8 ± 2.0) × 10⁻³		k_cat/K_M (s⁻¹ µM⁻¹)		(3.3 ± 0.7) × 10⁻²
MnCl₂	K_M (µM)	2.0 ± 0.3	(1.64 ± 0.01) × 10³	MnCl₂	K_M (µM)		422 ± 67
	V_max (nM s⁻¹)	416 ± 13	290 ± 12		V_max (nM s⁻¹)		421 ± 18
	k_cat/K_M (s⁻¹ µM⁻¹)	41 ± 12	(4.8 ± 0.5) × 10⁻⁴		k_cat/K_M (s⁻¹ µM⁻¹)		(2.5 ± 0.5) × 10⁻³
	k_cat (s⁻¹)	81 ± 11	0.78 ± 0.08		k_cat (s⁻¹)		1.04 ± 0.05

Reaction mixtures contain 10 mM MgCl₂ or MnCl₂. See ‘Methods’ for assay details. NaHCO₃ (100 mM, ~2.3 mM CO₂ at pH 8)²⁸ was used as a CO₂ source for the conversion of 2OG to isocitrate by wt IDH1. Due to the detection limit, K_Ms could not be determined accurately for <10 µM NADP⁺ or NADPH. See Supplementary Figs. S2 and S3 for kinetic plots. Calculated K_M and V_max values are reported as mean ± SEM, n = 3 technical replicates.
^aNot determined due to conversion without added MgCl₂.
^bNot measured due to the formation of insoluble MnCO₃ from MnCl₂ and NaHCO₃.

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