Nickel-induced substrate inhibition of bovine liver glutamate dehydrogenase.
The effects of nickel ions on reductive amination and oxidative deamination activities of bovine liver glutamate dehydrogenase (GDH) were examined kinetically by UV spectroscopy, at 27 degrees C, using 50 mM Tris, pH 7.8, containing 0.1 M NaCl. Kinetic analysis of the data obtained by varying NADH concentration indicated strong inhibition, presumably due to binding of the coenzyme to the regulatory site. In contrast, almost no inhibition was observed in the forward reaction. The fact that nickel ions have the capacity to enhance binding of NADH to the enzyme was confirmed by an electrochemical method using a modified glassy carbon electrode. Use of NADPH instead of NADH showed only a weak substrate inhibition, presumably related to lower affinity of NADPH for binding to the regulatory site. Lineweaver-Burk plots with respect to alpha-ketoglutarate and ammonium ions indicated substrate and competitive inhibition patterns in the presence of nickel ions, respectively. ADP at 0.2 mM concentration protected inhibition caused by nickel. These observations are explained in terms of formation of a nickel-NADH complex with a higher affinity for binding to the regulatory site in GDH, as compared with the situation where nickel is not present. Such effects may be important for regulation of GDH and other NADH-utilizing enzymes.
Ghobadi
S
S
Institute of Biochemistry and Biophysics, University of Tehran, Iran.
Nemat-Gorgani
M
M
Golabi
S M
SM
Zare
H R
HR
Moosavi-Movahedi
A A
AA
D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors*; D007656:Ketoglutaric Acids / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism / Q000494:pharmacology*; D009532:Nickel / Q000494:pharmacology*; D013379:Substrate Specificity
D007656:Ketoglutaric Acids; D009243:NAD; D000641:Ammonia; D009532:Nickel; D005969:Glutamate Dehydrogenase