Kinetic characterization of human hydroxyacid–oxoacid transhydrogenase: Relevance toD‐2‐hydroxyglutaric and γ‐hydroxybutyric acidurias

Abstract

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.