The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase

Abstract

The proteins glutamate dehydrogenase (GDH) and mitoNEET are both targets of drug development efforts to treat metabolic disorders, cancer, and neurodegenerative diseases. However, these two proteins differ starkly in the current knowledge about ligand binding sites. MitoNEET is a [2Fe-2S]-containing protein with no obvious binding site for small ligands observed in its crystal structures. In contrast, GDH is known to have a variety of ligands at multiple allosteric sites thereby leading to complex regulation in activity. In fact, while GDH can utilize either NAD(H) or NADP(H) for catalysis at the active site, only NAD(H) binds at a regulatory site to inhibit GDH activity. Previously, we found that mitoNEET forms a covalent bond with GDH in vitro and increases the catalytic activity of the enzyme. In this study we evaluated the effects of mitoNEET binding on the allosteric control of GDH conferred by inhibitors. We examined all effectors using NAD or NADP as the coenzyme to determine allosteric linkage by the NAD-binding regulatory site. We found that GDH activity, in the presence of the inhibitory palmitoyl-CoA and EGCG, can be rescued by mitoNEET, regardless of the coenzyme used. This suggests that mitoNEET rescues GDH by stabilizing the open conformation.

Evolutionary Changes in Primate Glutamate Dehydrogenases 1 and 2 Influence the Protein Regulation by Ligands, Targeting and Posttranslational Modifications

“…Notably, an integral mitochondrial membrane [2Fe-2S]-containing protein mitoNEET has been discovered to form a disulfide bond between mitoNEET Cys84 residue and a GDH residue corresponding to human Cys376, which increases the GDH catalytic activity in vitro [96]. Additionally, mitoNEET can significantly decrease GDH inhibition by palmitoyl-CoA and epigallocatechin gallate [97]. GLUD1 phosphorylated at Ser384 has been shown to interact with nuclear factor RelA and with serine kinase IKKβ in human cancer cells [86].…”