Quaternary structure and activity of glutamate dehydrogenase are regulated by reversible S‐palmitoylation and mitochondrial acyl‐protein thioesterases

Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that links amino acid and carbohydrate metabolism through the reversible conversion of glutamate to α-ketoglutarate. GDH functions as a hexamer and is subject to extensive allosteric regulation. Here, we demonstrate that GDH undergoes auto-palmitoylation in the presence of palmitoyl-CoA, accompanied by concentration-dependent inhibition of both reductive amination and oxidative deamination. Acyl-PEG exchange assays indicated predominantly one palmitoyl modification per GDH subunit, while mass spectrometry identified Cys55, Cys115, and Cys197 as putative modification sites. Blue native PAGE revealed that palmitoylation promotes dissociation of GDH hexamers into species consistent with dimers. Treatment with acyl-protein thioesterase 1 (APT1) restored both hexameric assembly and enzymatic activity, whereas α/β-hydrolase domain-containing protein 10 (ABHD10) restored activity less efficiently under the conditions tested. Acyl-resin-assisted capture also detected endogenous S-acylated GDH in three human cell lines, and treatment with the APT1 inhibitor ML348 increased GDH S-acylation in HepG2 cells. Together, these findings identify reversible S-palmitoylation as a mechanism regulating GDH assembly and activity, with the potential to couple mitochondrial lipid metabolism to amino acid catabolism.