Kinetic characterization of human hydroxyacid–oxoacid transhydrogenase: Relevance toD-2-hydroxyglutaric and γ-hydroxybutyric acidurias
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We investigated the presence of hydroxyacid–oxoacid transhydrogenase (HOT), which catalyses the cofactor-independent conversion of γ-hydroxybutyrate (GHB) to succinic semialdehyde coupled to reduction of 2-ketoglutarate (2-KG) to D-2-hydroxyglutarate (D-2-HG), in human liver extracts employing [2H6]GHB and 2-KG as substrates. We measured incorporation of 2H in D-[2H]2-HG using GC-MS analyses, providing evidence for HOT activity in humans. Kinetic characterization of HOT was undertaken in forward and reverse directions. We employed [2H6]GHB and [2H4]2-KG as cosubstrates in order to develop a HOT activity assay in cultured human fibroblasts derived from patients with D-2-hydroxyglutaric aciduria. HOT activity was quantified in this system by the measurement of D-[2H5]2-HG production. Fibroblasts derived from patients with D-2-hydroxyglutaric aciduria showed normal HOT activities. Our results provide the first demonstration and preliminary kinetic characterization of HOT activity in human tissues.
KeywordsPublic Health Internal Medicine Reverse Direction Metabolic Disease Activity Assay
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- Gibson KM, Craigen W, Herman GE, et al (1993a) D-2-Hydroxyglutaric aciduria in a newborn with neurological abnormalities: a new neurometabolic disorder? J Inherit Metab Dis 16: 497–500.Google Scholar
- Gibson KM, ten Brink HJ, Schor DSM, et al (1993b) Stable isotope dilution analysis of D- and L-2-hydroxyglutaric acid: application to the detection and prenatal diagnosis of D- and L-2-hydroxyglutaric acidemias. Pediatr Res 34: 277–280.Google Scholar
- Kaufman EE, Nelson T, Fales MF, et al (1988a) Isolation and characterization of a hydroxyacid-oxoacid transhydrogenase from rat kidney mitochondria. J Biol Chem 263: 16872–16879.Google Scholar
- Kaufman EE, Nelson T, Miller D, et al (1988b) Oxidation of gamma-hydroxybutyrate to succinic semialdehyde by a mitochondrial pyridine nucleotide-independent enzyme. J Neurochem 51: 1079–1084.Google Scholar