title: A tale of two subunits: how the neomorphic R132H IDH1 mutation enhances production of αHG.
abstract: Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.
authors.affiliation: Departments of Biological Reagents and Assay Development, Screening and Compound Profiling, Statistical Sciences, Computational and Structural Sciences, and Cancer Metabolism, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA.
authors.forename: Beth
authors.initials: B
authors.lastname: Pietrak
authors.forename: Huizhen
authors.initials: H
authors.lastname: Zhao
authors.forename: Hongwei
authors.initials: H
authors.lastname: Qi
authors.forename: Chad
authors.initials: C
authors.lastname: Quinn
authors.forename: Enoch
authors.initials: E
authors.lastname: Gao
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Boyer
authors.forename: Nestor
authors.initials: N
authors.lastname: Concha
authors.forename: Kristin
authors.initials: K
authors.lastname: Brown
authors.forename: Chaya
authors.initials: C
authors.lastname: Duraiswami
authors.forename: Richard
authors.initials: R
authors.lastname: Wooster
authors.forename: Sharon
authors.initials: S
authors.lastname: Sweitzer
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schwartz
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005977:Glutarates / Q000378:metabolism*; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D053719:Tandem Mass Spectrometry
chemical_list: D005977:Glutarates; C019417:alpha-hydroxyglutarate; D007521:Isocitrate Dehydrogenase; C543588:IDH1 protein, human