Kinetic characterization of human hydroxyacid-oxoacid transhydrogenase: relevance to D-2-hydroxyglutaric and gamma-hydroxybutyric acidurias.
We investigated the presence of hydroxyacid-oxoacid transhydrogenase (HOT), which catalyses the cofactor-independent conversion of gamma-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.
Struys
E A
EA
Metabolic Unit, Department of Clinical Chemistry, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. e.struys@vumc.nl
Verhoeven
N M
NM
Ten Brink
H J
HJ
Wickenhagen
W V
WV
Gibson
K M
KM
Jakobs
C
C
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D000592:Amino Acid Metabolism, Inborn Errors / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005977:Glutarates / Q000378:metabolism*; D006801:Humans; D006885:Hydroxybutyrates / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry; D024101:Mitochondrial Proteins; D013997:Time Factors
D005977:Glutarates; D006885:Hydroxybutyrates; D024101:Mitochondrial Proteins; C019417:alpha-hydroxyglutarate; C111420:4-hydroxybutyric acid; D000429:Alcohol Oxidoreductases; C072878:hydroxyacid-oxoacid transhydrogenase