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 pM and 325 pM, 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 A @ = + 3.3 kJ mol-' (polypeptide chain)-' 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 slow.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.The enzymatic activity of beef liver glutamate dehydrogenase is modulated by purine nucleotides in a complex fashion [5,6]. ADP is known to be an activator above pH 7 whereas GTP strongly inhibits the enzymatic reaction under This work has...

The Glutamate Dehydrogenase Pathway and Its Roles in Cell and Tissue Biology in Health and Disease

“…These include purine nucleotides (ADP, ATP and GTP) [34,42], NADH [34], L-leucine [43], palmityl CoA [44], spermidine [45,46], steroid hormones [47,48], neuroleptic drugs [49,50,51] and the green tea polyphenol epigallocatechin gallate (EGCG) [46,52]. Recently, the purpurin analogue R162 (2-allyl-1-hydroxy-9,10-anthraquinone) was identified as a potent inhibitor of GDH1 [3].…”

Thermal aggregation of a model allosteric protein in different conformational states

“…GDH is subject to allosteric regulation by l-leucine, ADP, GTP, and a number of other nucleotides and hormones [34,35]. Also, each of the six subunits in the enzyme hexamer has two binding sites for NAD + or NADH [36].…”

The structure and allosteric regulation of mammalian glutamate dehydrogenase

“…In contrast, GTP is a potent inhibitor and is thought to act by stabilizing abortive complexes [5]. GTP binding is antagonized by phosphate [6] and ADP [7], but is proposed to be synergistic with NADH bound in the non-catalytic site [6]. Finally, ADP and GTP bind in an antagonistic manner [7] either due to steric competition or due to competing effects on abortive complex formation.…”

“…GTP binding is antagonized by phosphate [6] and ADP [7], but is proposed to be synergistic with NADH bound in the non-catalytic site [6]. Finally, ADP and GTP bind in an antagonistic manner [7] either due to steric competition or due to competing effects on abortive complex formation. As discussed below, it is likely that these regulators act by modulating the enzyme dynamics [8, 9].…”