UC San Diego

Changes in cancer cell metabolism was described by Otto Warburg in the early 1900s, but it was not until relatively recent that specific metabolic enzymes can drive tumorigenesis and tumor growth. Mutations in isocitrate dehydrogenase 1 (IDH1), more specific at residue 132, were linked to brain cancer in 2008. IDH1 is a metabolic enzyme responsible for converting isocitrate to α-ketoglutarate (αKG) producing NADPH. The latter two metabolites are crucial for the cell to thrive healthy. However, when IDH1 is mutated, they lose their normal catalysis, while gaining a new activity by converting αKG to D-2-hydroxyglutarte (D2HG), depleting NADPH. D2HG promotes oncogenesis by inhibiting enzymes involved in gene regulation. The goal of this dissertation is to understand the structure-function relationship of tumor-relevant IDH1 mutants using, primarly, kinetic approaches as well as other biophysical methods.

Steady-state kinetics was used to characterize the catalytic profile of several tumor and non-tumor relevant IDH1 mutants. This work highlights a wide range in catalytic efficiency within mutants as well as providing the foundation to predict disease severity depending on the mutant seen in patients. Inhibition studies revealed that small molecule inhibitor affinity varies widely among mutant IDH1 forms, providing a solid foundation for predicting how patients may responde to therapeutics as well as possible resistance mutation that may arise from target therapy. Lastly, a comprehensive catalytic cycle for D2HG production by tumor-relevant IDH1 mutants was stablished using pre-steady-state kinetics as well as biochemical and biophysical methodologies. This work determined a wide variation in intrinsic rate constants associated to substrate binding, thus revealing key kinetic features driving the neomorphic reaction and highlights kinetic features that explain mechanistic differences among IDH1 mutants.