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Kinetic characterization of human hydroxyacid–oxoacid transhydrogenase: Relevance to D ‐2‐hydroxyglutaric and γ‐hydroxybutyric acidurias
Author(s) -
Struys E. A.,
Verhoeven N. M.,
Ten Brink H. J.,
Wickenhagen W. V.,
Gibson K. M.,
Jakobs C.
Publication year - 2005
Publication title -
journal of inherited metabolic disease
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 102
eISSN - 1573-2665
pISSN - 0141-8955
DOI - 10.1007/s10545-005-0114-x
Subject(s) - chemistry , biochemistry , enzyme , cofactor , stereochemistry
Summary 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 [ 2 H 6 ]GHB and 2‐KG as substrates. We measured incorporation of 2 H in D ‐[ 2 H]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 [ 2 H 6 ]GHB and [ 2 H 4 ]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 ‐[ 2 H 5 ]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.