The effect of pyridoxal phosphate modification on the catalytic and regulatory properties of bovine liver glutamate dehydrogenase.

The kinetic properties of bovine liver glutamate dehydrogenase have been examined as a function of pyridoxal phosphate modification, this reagent having been previously shown to react specifically with lysine-97 of the tentative sequence proposed by Smith et al. ((1970) Proc. Nat. Acad. Sci. U. S. A. 67, 724). It is found that partial modification (two to three groups per six chains in the active enzyme) leads to almost complete loss of excess NADH inhibition. Modification also leads to an increase in the inhibition constant for GTP as well as a progressive loss of activity. The nature of these changes suggest strong subunit-subunit interaction with respect to some of the regulatory properties of the enzyme. The altered inhibition constant for GTP is apparently a consequence of a change in the rate of a coenzyme-GTP-induced conformational change. Loss of activity after pyridoxal phosphate modification may not be complete since some residual activity always remains. Furthermore, modification has a slight activating effect on activity when the enzyme is assayed in the presence of excess GTP. It is concluded that, although lysine-97 is an important residue in the catalytic reaction, it may not be essential for activity. Prior modification of lysine-428 with trinitrobenzenesulfonate, which also results in loss of excess NADH inhibition, does not affect the rate or extent of pyridoxal phosphate modification.