Investigation of the effects of crosslinking glutamate dehydrogenase with dimethyl pimelimidate.
Chemical crosslinking with dimethyl pimelimidate has been used to examine the quaternary structure and conformational mobility of bovine liver glutamate dehydrogenase. Crosslinking patterns are shown to be consistent with either a stacked or staggered dimer of trimers structure of the hexamer. Crosslinking in the absence of coligands results in a small loss of activity but an almost complete loss of GTP inhibitory effects. Protection experiments show that the active site can be protected by a variety of ligand combinations, and that the loss of GTP inhibition is protected by several complexes containing either NADH or NADPH, indicating that the second coenzyme site per subunit (which preferentially binds NADH) is not involved in the protection process. A significant loss of ADP activation occurs during crosslinking which is not protected against by any combination of protecting ligands tried, including those which involve second coenzyme site binding, showing that the ADP site is functionally distinct from the GTP site and from the second coenzyme binding site. Crosslinking in the presence of protecting ligands gives similar gel patterns to those obtained in the absence of protection. Affinity chromatography experiments show that the crosslinked enzyme still binds GTP despite the loss of GTP inhibition, and hysteresis experiments show that the second coenzyme site is left functional if protected with either coenzyme. A model is presented where crosslinking affects the conformational linkage between various ligand binding sites involved in GTP inhibition rather than the sites themselves.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003720:Densitometry; D004789:Enzyme Activation / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007096:Imidoesters / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
D007096:Imidoesters; D046911:Macromolecular Substances; D009243:NAD; C029326:dimethyl pimelimidate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase