The mechanism of activation of glutamate dehydrogenase-catalyzed reactions by two different, cooperatively bound activators.

The activation of NADHand NADPH-dependent glutamate dehydrogenase-catalyzed reactions appears to be similar for two structurally different compounds, ADP and any of a series of monocarboxylic L-a-amino acids. ADP and L-leucine have identical effects on a large variety of glutamate dehydrogenase-catalyzed reactions. Transient state studies show that L-leucine increases the rate of release of NADPH from an enzyme-NADPH complex in a manner similar to that of ADP. The dissociation constant for the glutamate dehydrogenaseNADPH complex as measured by difference spectroscopy is increased by leucine, indicating that leucine interferes with this mode of NADPH binding. It is shown that the dissociation constant for this leucine complex which interferes with a mode of NADPH binding is equal to the reported dissociation constant for the binary enzyme-leucine complex. The steady state kinetic activation constant which can be obtained by extrapolation to infinitely dilute substrate concentrations is equal to the dissociation constant of the binary enzyme-leutine complex. Since the tight binding of NADPH measured spectroscopically in the 340 nm region appears to be involved in a rate-limiting step, the mechanism of activation of ar-ketoglutarate reduction by ADP and monocarboxylic L-o-amino acids may be to weaken this NADPH binding mode. Although kinetic activation data and the binding interactions with NADPH are similar for ADP and L-leucine, each activator enhances the binding of the other and, together, they have a slightly additive activation. Spectroscopic data presented in this paper indicate that the two types of activators have spatially separate binding sites on the enzyme and interact in a positive manner to enhance the binding of each other (i.e. positive cooperativity).