Mutations in isocitrate dehydrogenase 1 (IDH1) are the drivers of most low-grade gliomas, secondary glioblastomas, and many cases of chondrosarcoma and acute myeloid leukemia. These mutations reduce the ability of IDH1 to oxidize isocitrate to α-ketoglutarate (αKG) and can cause the enzyme to acquire a neomorphic activity in which it converts αKG to D-2-hydroxyglutarate (D2HG). In previous work, the α10 regulatory domain of IDH1 was shown to play a role in substrate and inhibitor binding. To further investigate the role of this region of the protein, mutations were designed and introduced to either stabilize or destabilize this domain. We also designed a mutant of IDH1 that had the regulatory domain of IDH2 which we believed would exhibit kinetic characteristics similar to those of IDH2. Using UV-Vis spectroscopy, we determined the kinetic parameters of these mutants. We also used hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) to closely examine the regions of IDH1 that are involved in substrate binding. We find that mutant IDH1 is unable to withstand any mutational burden in the α10 regulatory domain. We also detail regions within the structure of IDH1 that proved to be dynamic upon addition of various substrates. These findings will give new insights into regions of IDH1 that should be further examined for use in drug design for cancers driven by IDH1 mutation.