Skip to main content
. Author manuscript; available in PMC: 2022 Sep 16.
Published in final edited form as: Mol Cell. 2021 Jul 20;81(18):3833–3847.e11. doi: 10.1016/j.molcel.2021.06.027

Figure 3. K413-acetylation attenuates substrate (α -KG) and co-factor (NADPH) binding to mIDH2.

Figure 3.

(A-B) Purified rIDH2 R140Q variants were either pre-treated with cobB followed by incubation with rACAT1 (A) or treated with rSIRT3 (B), prior to 14C-labeled α-KG binding assay.

(C-D) Vmax and Km of rIDH2 R140Q variants were measured using purified rIDH2 R140Q proteins that were either pre-treated with cobB followed by incubation with rACAT1 (C) or treated with rSIRT3 (D), followed by mIDH2 enzyme activity assay in the presence of increasing concentrations of either substrate α-KG (left panels) or cofactor NADPH (right panels). Vmax and Km values of each treated group were calculated (lower panels) and plotted (upper panels). Please note that the comparable Vmax values of IDH2 R140Q and acetyl-deficient R140Q/K413R are due to the condition that purified mIDH2 proteins were pre-treated with cobB to remove intrinsic lysine acetylation obtained in bacteria prior to rACAT1 incubation.

The error bars represent mean values ±SD from three replicates of each sample (***: p<0.001; ns: not significant); Data are mean ± SD; p values were obtained by a two-tailed Student’s t-test.