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Biochemical Characterization of Heterodimeric Isocitrate Dehydrogenase I Using a Novel High‐throughput Mass Spectrometry‐based Assay

In several types of human tumors, a gain‐of‐function mutation within a single allele of isocitrate dehydrogenase 1 (IDH1) results in the production of a known oncometabolite, 2‐hydroxyglutarate (2‐HG). IDH1 is enzymatically active as a dimer, and both homodimeric and wt:mutant heterodimeric proteins are produced in cells. Since the heterodimer is comprised of both the mutant and wild‐type subunits, it therefore catalyzes both the canonical (a‐ketoglutarate generating) and the neomorphic (2‐HG generating) reactions. Traditionally, IDH1 assays monitor the reduction or oxidation of NADP/H, a common co‐factor substrate for both the mutant and wild‐type subunits. This, in conjunction with an extremely low predicted NADPH Km, greatly complicates the kinetic characterization of heterodimeric IDH1. Here we present a novel mass‐spectrometry‐based IDH1 assay. Not only are we able to selectively monitor 2‐HG production by the mutant subunit, but incorporation of a unique NADPH regeneration system allows us to study the reaction at nanomolar NADPH concentrations. Using this platform we determined that the IDH1 heterodimer exhibits a significantly lower Km for both a‐KG (314 μM) and NADPH (26 nM) than the mutant homodimeric protein (a‐KG Km = 2800 μM and NADPH Km = 173 nM). To further characterize the heterodimer and determine if the wild‐type subunit catalyzes production of NADP+ and isocitrate, we assessed the activity of a catalytically impaired wild‐type subunit (Y139D) and also monitored the heterodimeric reaction by NMR. Our findings indicate that under the conditions of our assay, the wild‐type subunit makes minimal contributions to total NADP+ production.