Kinetic studies on the mechanism of the action of ADP on the glutamate dehydrogenase reaction.

ADP is known to activate the glutamate dehydrogenase (GluDH EC 1.4.1.3) reaction above pH 7 [2,3] whereas below this pH ADP is inhibitory. Therefore, it has been postulated that at low pH values, ADP instead of binding to an activator site binds to the inhibitor site which at high pH values is used by GTP [4] Since NAD’ shows a strong self-activating effect [5], the activation by ADP is dependent on NAD’ concentration. The extrapolation of previous experimental data suggested that ADP was inhibitory at high NAD’ concentrations [2] However, before a general model for the ADP effect can be constructed, experiments must be performed to measure the ADP effect at low NAD’ concentrations and to make a detailed investigation of the pH dependence of this reaction. The data presented in this paper show that at all pH values, ADP has two effects: i) Increase of Vmax with a simultaneous increase of the Michaelis constant for NAD’. This effect causes activation at high and inhibition at very low NAD’ concentrations. ii) Competitive inhibition with an inhibitor constant of about 6 mM which leads to an even further inhibition at low NAD’ concentrations. The ratio of inhibition to activation increases with decreasing pH and increasing phosphate concentration. It is shown that all the observed effects can be explained on the basis of a single adenine binding subsite of the coenzyme binding site.