Active site remodeling in IDH1 mutants drives distinct kinetic and potential resistance mechanisms.

Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved enzyme that converts isocitrate to α-ketoglutarate (αKG). IDH1 mutations affecting residue R132 drive tumor formation, and several FDA-approved mutant IDH1 inhibitors are available. Most mutants cannot catalyze the conventional reaction, but gain a neomorphic activity that produces D-2-hydroxyglutarate (D2HG). We previously reported that IDH1 R132Q uniquely preserves conventional activity while catalyzing robust D2HG production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding, hydride transfer, and prevention of inhibitor binding. When binding isocitrate, IDH1 R132Q exhibited a closed conformation with features consistent with decreased catalysis versus WT. Binding neomorphic reaction substrates resulted in semi-closure of IDH1 R132Q with profoundly limited active site solvent accessibility, and featured a distinct αKG binding pocket compared to R132H. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions that can improve inhibitor selectivity and drive resistance.