Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5'-phosphate.
The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology
D007656:Ketoglutaric Acids; D009243:NAD; D011732:Pyridoxal Phosphate; D005969:Glutamate Dehydrogenase; D008239:Lysine