Evidence from Circular-dichroism Studies for a Conformational Change in the Ternary Complex Enzyme-(oxidized Nicotinamide- Adenine Dinucleotide)-glutarate

1. Computer averaging of multiple scans was used to refine the circular dichroism spectrum of bovine liver glutamate dehydrogenase, revealing well-defined structure in the aromatic region. 2. The circular dichroism ofNAD+ bound to glutamate dehydrogenase is strongly negative at 260nm, probably owing to immobilization of the adenosine moiety. Loss of the characteristic adenine-nicotinamide interaction suggests that the coenzyme is bound in an unstacked conformation. 3. Glutarateand succinate, substrate analogues that are both inhibitors competitive with glutamate, do not significantly perturb the circulardichroism spectrum of the enzyme in the absence ofNAD+. 4. In the presence ofNAD+, 150mM-succinate decreases the negative circular dichroism corresponding to bound coenzyme, but does not affect the protein circular dichroism. However, 150mm-glutarate causes profound alterations of the circular-dichroism spectra ofthe bound NAD+ and of the enzyme, indicative of a protein conformational change. This direct evidence of conformational change specifically promoted by C5 dicarboxylates confirms the previous inference from protection studies. 5. The conformational change is discussed in relation to the allosteric mechanism of glutamate dehydrogenase.