Magnetic-resonance studies of the geometry of bound substrate, coenzyme and activator on bovine-liver glutamate dehydrogenase.
ADP and ATP with a spin-label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin-label at a site in the active centre[Andree and Zantema, (1978) Biochemistry, 17, 778--783]. The spin-labelled activators interact with ketone spin-labelled glutamate dehydrogenase in the same way as with native glutamate dehydrogenase relative to the activator site, but show a stronger binding to the coenzyme site. Upon binding to the coenzyme site a spin-spin interaction between the ketone spin-label and the spin-labelled activators is observed. Nuclear magnetic resonance studies of the linewidth of 2-oxoglutarate and NADP+ bound to their functional sites on glutamate dehydrogenase without and with spin-labels result in distances between the ligand nuclei and the spin-labels. The results show that NADP+ binds in an open conformation consistent with the conformation in other dehydrogenases. The activator ADP binds in the neighbourhood of the active centre, but with very little or no overlap with the coenzyme site.
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005609:Free Radicals; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D066298:In Vitro Techniques; D007656:Ketoglutaric Acids / Q000378:metabolism; D007659:Ketones; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D013113:Spin Labels
D005609:Free Radicals; D007656:Ketoglutaric Acids; D007659:Ketones; D013113:Spin Labels; D009249:NADP; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005969:Glutamate Dehydrogenase