Electron spin resonance and nuclear relaxation studies on spin-labeled glutamate dehydrogenase.
The reaction of glutamate dehydrogenase with two different stable nitroxides (spin labels) is reported. The two compounds contain a carbonyl and an iodoacetamide group as their reactive parts. The carbonyl compound inactivates the enzyme by the formation of a 1:1 covalent complex after NaBH4 reduction of an intermediate Schiff's base. Evidence indicates that the enzyme is modified at lysine-126 in the active site. The electron spin resonance (ESR) spectrum of spin-labeled enzyme indicates a high degree of immobilization of the nitroxide. The binding of reduced coenzyme NADPH is reflected by a change (immobilization) of the ESR spectrum. Nuclear relaxation of bound substrate, oxidized coenzyme, and inhibitor by the paramagnetic group is observed. This shows the existence of a binding site for these compounds close to the active site. The distances of selected protons of the binding ligands to the nitroxide are calculated. The iodoacetamide spin label reacts with several groups, one of which is not a sulfhydryl. The reaction of this particular group causes inactivation of the enzyme. Protection against this inactivation could be achieved with certain ligands. Only enzyme that was spin labeled without such protection caused paramagnetic relaxation of bound substrate and coenzyme.
D000495:Allosteric Site / Q000187:drug effects; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013113:Spin Labels; D013696:Temperature
D007656:Ketoglutaric Acids; D013113:Spin Labels; D009243:NAD; D009249:NADP; D011732:Pyridoxal Phosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase