Description
Changes in cancer cell metabolism were first described by Otto Warburg in the early 1900s, but it was not until relatively recently that specific metabolic enzymes were shown to drive tumorigenesis and tumor growth. An example of a metabolic enzyme driving tumor formation is isocitrate dehydrogenase 1. Mutations in isocitrate dehydrogenase 1 (IDH1), more specificly at residue 132, were first linked to brain cancer in 2008. IDH1 is responsible for converting isocitrate and NADP+ to α-ketoglutarate (αKG) and NADPH. The latter two metabolites are crucial for many metabolic cell processes. However, when IDH1 is mutated, this enzyme cannot perform its normal catalysis, but instead gains a neomorphic activity of converting αKG to D-2-hydroxyglutarte (D2HG), depleting NADPH. D2HG promotes oncogenesis by inhibiting αKG -dependent enzymes, including those involved in gene regulation. The goal of this dissertation is to understand the structure-function relationship of tumor-relevant IDH1 mutants using, kinetic and other biophysical methods. Steady-state kinetics was used to characterize the catalytic profile of several tumor-relevant and experimental IDH1 mutants. These mutants had a wide range in catalytic efficiency, providing a foundation for predicting disease severity depending on the mutant seen in patients. Inhibition studies revealed that small molecule inhibitor affinity varied widely among mutant IDH1 forms, providing a solid foundation for predicting how patients may respond to therapeutics as well as possible resistance mutation that may arise from targeted therapy. Lastly, a comprehensive catalytic cycle for D2HG production by tumor-relevant IDH1 mutants was established 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 ke kinetic features driving the neomorphic reaction. This highlighted kinetic features that may help explain the mechanistic differences observed among IDH1 mutants.
