Cancer-associated isocitrate dehydrogenase mutations inactivate NADPH-dependent reductive carboxylation

Abstract

Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP(+)-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and the NADPH/CO(2)-dependent reductive carboxylation of αKG to ICT. Reductive carboxylation by IDH1 was potently inhibited by NADP(+) and, to a lesser extent, by ICT. IDH1 and IDH2 with cancer-associated mutations at the active site arginines were unable to carry out the reductive carboxylation of αKG. These mutants were also defective in ICT decarboxylation and converted αKG to 2-hydroxyglutarate using NADPH. These mutant proteins were thus defective in both of the normal reactions of IDH. Biochemical analysis of heterodimers between wild-type and mutant IDH1 subunits showed that the mutant subunit did not inactivate reductive carboxylation by the wild-type subunit. Cells expressing the mutant IDH are thus deficient in their capacity for reductive carboxylation and may be compromised in their ability to produce acetyl-CoA under hypoxia or when mitochondrial function is otherwise impaired.

FIGURE 1.

Regulation of IDH1 reductive carboxylation by NADP⁺. Reaction rates were determined by following the decrease in NADPH fluorescence as a function of time as described under “Experimental Procedures.” A, the time course for the carboxylation of αKG by IDH1 was linear for only the first few minutes of the assay. Initial rates were calculated from the data in the first 3 min of the reaction. B, inhibition of IDH1 reductive carboxylation of αKG by NADP⁺ using the spectrofluorometric assay. C, inhibition of IDH1 reductive carboxylation by ICT using the spectrofluorometric assay. The IDH1 specific activity (100% activity) in these experiments was 142 pmol/min/mg, and data points were obtained in triplicate.

FIGURE 2.

Reductive formation of ICT and 2HG by wild-type and mutant IDH1 proteins. A, comparison of the radiochemical ([¹⁴C]αKG conversion to ICT and 2HG) assay for IDH1 in the presence and absence of a Glc-6-P dehydrogenase (G6P DH) NADPH-regenerating system. B, analysis of the products formed by IDH1 homodimers (WT/WT), IDH1^WT/R132H heterodimers (WT/R132H), and IDH1(R132H) homodimers (R132H/R132H) using the radiochemical assay with the NADPH-regenerating system to simultaneously detect [¹⁴C]ICT and [¹⁴C]2HG by thin-layer chromatography as described under “Experimental Procedures.”

FIGURE 3.

Production of ICT and 2HG by wild-type and mutant IDH1 proteins. A, representative time course for the formation of ICT by IDH1 homodimers (WT/WT; ●), IDH1(R132H) homodimers (R132H/R132H; ○), and IDH1^WT/R132H heterodimers (WT/R132H; ■). Reaction rates slowed with time due to product inhibition by ICT. B, representative time course for the formation of 2HG by the three types of IDH1 proteins. C, the IDH1 specific activities for individual IDH1 proteins was determined in at least two different lots of individually purified proteins using the radiochemical assay to detect both [¹⁴C]ICT and [¹⁴C]2HG formation.

FIGURE 4.

NADP⁺, ICT, and 2HG regulation of IDH1 and IDH1(R132H). A, NADP⁺ inhibition of NADPH oxidation by IDH1(R132H) homodimers (R132H/R132H; ○) and IDH1^WT/R132H heterodimers (WT/R132H; ■). B, ICT regulation of NADPH oxidation by IDH1(R132H) homodimers (○) and IDH1^WT/R132H heterodimers (■). C, 2HG inhibition of NADPH oxidation by IDH1 homodimers (WT/WT; ●), IDH1(R132H) homodimers (○), and IDH1^WT/R132H heterodimers (■). The spectrofluorometric assay following αKG-dependent NADPH oxidation was used in these experiments. Means ± S.E. of triplicate measurements are plotted.