title: The binding of oxidized coenzymes by glutamate dehydrogenase and the effects of glutarate and purine nucleotides.
abstract: 1. The binding of NAD(+) and NADP(+) to glutamate dehydrogenase has been studied in sodium phosphate buffer, pH7.0, by equilibrium dialysis. Approximate values for the dissociation constants are 0.47 and 2.5mm respectively. For NAD(+) the value agrees with that estimated from initial-rate results. 2. In the presence of the substrate analogue glutarate both coenzymes are bound more firmly, and there is one active centre per enzyme subunit. The binding results cannot be described in terms of independent and identical active centres, and binding is stronger at low coenzyme concentrations than at high concentrations. Either the six subunits of the oligomer are not identical or there are negative interactions between them in the binding of coenzymes in ternary complexes with glutarate. The latter explanation is favoured. 3. The binding studies support the conclusions drawn from earlier kinetic studies of the glutamate reaction. 4. ADP and GTP respectively decrease and increase the affinity of the enzyme for NAD(+) and NADP(+), in both the presence and absence of glutarate. The negative binding interactions in the presence of glutarate are abolished by ADP, which decreases the affinity for the coenzymes at low concentrations of the latter. 5. In the presence of glutarate, GTP and NAD(+) or NADP(+), the association of enzyme oligomers is prevented, and the solubility of the enzyme is decreased; the complex of enzyme and ligands readily crystallizes. 6. The results are discussed in relation to earlier kinetic studies.
mesh_terms: D000244:Adenosine Diphosphate; D001665:Binding Sites; D003956:Dialysis; D005969:Glutamate Dehydrogenase; D005977:Glutarates; D006160:Guanosine Triphosphate; D007700:Kinetics; D009243:NAD; D009249:NADP; D013053:Spectrophotometry; D014461:Ultracentrifugation
chemical_list: D005977:Glutarates; D009243:NAD; D009249:NADP; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase