Studies of Glutamate Dehydrogenase

Abstract

1 Equilibrium dialyses have shown tighter binding of GTP to glutamate dehydrogenase in triethanolamine than in phosphate buffer, as characterized by dissociation constants of 35 μM and 325 μM, respectively. If competition between GTP and phosphate occurs, a dissociation constant of 13 mM can be calculated for the binding of phosphate. Binding of ADP, however, is not changed when phosphate buffer is replaced by triethanolamine buffer. 2 The binding of GTP and ADP to the enzyme as well as their mutual interaction in the bound state has also been examined by equilibrium dialyses. Both nucleotides bind simultaneously but with increased dissociation constants. For this negative interaction of ADP and GTP, a coupling energy of ΔG0c=+ 3.3 kJ mol−1 (polypeptide chain)−1 can be calculated. The total number of sites occupied by the two effectors in the ternary complex is identical to the sum of binding sites for the two nucleotides in their respective binary complexes. Thus separate although interacting sites exist for ADP and GTP. 3 Nucleotide binding within the ternary glutamate dehydrogenase-NADPH-GTP complex has been correlated with ligand-induced dissociation of the enzyme to the unimer (the functional subunit comprising six polypeptide chains). Equilibrium dialyses revealed curved Scatchard plots indicating positive cooperative formation of the ternary complex from both binary enzyme-nucleotide complexes. In addition to indicating decreased dissociation constants of the two nucleotides, the binding curves reflect the ligand-induced dissociation of the enzyme into the unimer. Sedimentation analyses of the latter process show close agreement between the degree of ternary complex formation and the extent of dissociation of the enzyme. Two defined states of the enzyme can be distinguished. One state exists as the binary enzyme · nucleotide complex with an unchanged association equilibrium; the other state is completely dissociated into unimers which are saturated with both nucleotides, GTP and NADPH. The exchange between these two states is low. 4 The influence of ADP on the ternary enzyme-NADPH-GTP complex has been studied with respect to both the nucleotide binding and to the association equilibrium of the enzyme. ADP strongly decreases the affinity of the enzyme for both NADPH and GTP and, in addition, restores the ability of the enzyme to associate. The latter process appears to be complete after two or three of the total of six ADP-binding sites per unimer are saturated. The quaternary complex, enzyme-NADPH-GTP-ADP, stabilizes the associated state of the enzyme. However, at high concentration of GTP and ADP, the dissociation constant for NADPH binding to the enzyme is the same as in the enzyme · NADPH binary complex. Thus the effects of two purine nucleotides cancel each other.