Glutamate Dehydrogenase Concentration as a Determinant in the Effect of Purine Nucleotides on Enzymatic Activity

The properties of bovine liver glutamate dehydrogenase with respect to the effects of purine nucleotides have been examined over a wide range of enzyme concentrations. Kinetic studies, with enzyme concentration up to 1 mg per ml have been performed with both guanosine triphosphate and diphosphate as inhibitors and adenosine diphosphate as an activator of the enzyme. Direct binding experiments have been performed to examine GTP binding in the presence of reduced diphosphopyridine nucleotide over the range of 0.1 to 14.7 mg per ml. Both the kinetic and direct binding studies support the conclusion that, in the presence of coenzyme, GTP and GDP bind less tightly to the associated polymeric form (which has a molecular weight of 1.6 to 2 x 106 and is formed as a result of a concentration-dependent association reaction) than they do to the monomeric form of the enzyme (molecular weight, 4 x 105). The preferential binding of these nucleotides to monomer gives rise to an apparent cooperative interaction between inhibitor sites (either GTP or GDP) at enzyme concentrations greater than 0.1 mg per ml. No such effects exist at lower enzyme levels (l0.01 mg per ml), presumably because no polymer is present. In contrast to these results, ADP, which activates the reaction, has been found in the presence of reduced pyridine nucleotide to bind preferentially to the polymer rather than to the monomer. It is postulated that DPNII and reduced triphosphopyridine nucleotide alone behave in the same manner as ADP. Experiments have been performed to evaluate the effect of both ADP and GTP (or GDP) simultaneously on the kinetic properties at high enzyme concentrations. Kinetic data and theory are presented for the situation where the enzyme concentration is larger than the dissociation constant for one of the ligands (that is, where the free and total ligand concentration cannot be considered to be equivalent) as is the case for inhibition by GTP. The theory is shown to be valid for a chemically modified glutamate dehydrogenase which is enzymatically normal in all respects but which cannot undergo association hat high enzyme concentrations. The theory presented here, however, is not adequate for native glutamate dehydrogenase presumably because of the preferential binding of GTP to the monomeric form of the enzyme. The consequence of preferential binding of purine nucleotides to different molecular weight species is discussed. It is noted that the ability of an enzyme to undergo association may be an important factor in relation to the effect of substrate or allosteric ligands on enzyme activity. Finally, it is shown by stop flow experiments that the specific activity of the enzyme in the absence of purine nucleotides is essentially independent of enzyme concentration.