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Doctor of Philosophy (PhD)




Mammals have three forms of isocitrate dehydrogenase (ICDH) that each catalyze the oxidative decarboxylation of isocitrate forming alpha-ketoglutarate (AKG), carbon dioxide and reduced pyridine nucleotide. The intra-mitochondrial, citric acid cycle enzyme is ICDH III that exists as a hetero tetramer and uses NAD+ as a substrate. ICDH I and ICDH II are both dimeric enzymes that reside in the cytosol and use NADP+. While, the precise function of the cytosolic ICDHs is not known, active site variants of both ICDH I and II cause a variety of cancers. The R132H variant of ICDH1 is one of the most common alterations. This form has an increased capacity to reduce the product AKG to form D-2 hydroxyglutarate (2HG) reforming NADP+. Interestingly the form of the enzyme with the greatest capacity for this chemistry is the hetero dimer of one intact and one variant copy of ICDH I. In this study we present transient state analysis of wild type, the R132H homodimer and the oncogenic hetero dimer. All three forms of the enzyme purify in an open, inactive form, that has NADPH tightly bound. The WT enzyme has a turnover number that decreases hyperbolically with increasing enzyme concentration, from a maximum of 18 s-1 to a minimum of 6 s-1 with a K½ of 0.9 µM. Incubation with D-ICT and Mg will induce the activation and release of precisely ½ of the bound NADPH indicating half-site reactivity. When mixed with NADP+, this IDCH1-Mg-ICT-NADPH50% complex forms NADPH at 210 s-1. This is followed by a second event of 30 s-1, which presumably is decarboxylation of oxalosuccinate. The crystal structure of the IDCH1-Mg-ICT-NADPH50% complex indicates one NADPH per dimer corresponding asymmetry. Curiously this structure also reveals a pseudo-tetramer interface of around 1000 Å2 that confines a helix and loop adjacent to the NADP binding site. This tetrameric form of ICDH I offers an explanation for the concentration dependence on the turnover number as a function of enzyme concentration. The oncogenic hetero tetramer also exhibits this type of behavior but diminishments in the native activity with increasing concentrations of enzyme are met with increases in the rate of 2HG formation under those same concentrations. This observation hints at the underlying mechanism for cellular proliferation.

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Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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