The allosteric mechanism of bovine liver glutamate dehydrogenase. Evidence from circular-dichroism studies for a conformational change in the ternary complex enzyme-(oxidized nicotinamide-adenine dinucleotide)-glutarate

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Abstract
Computer averaging of multiple scans was used to refine the circular dichroic spectrum of bovine liver glutamate dehydrogenase [EC 1.4.1.3], revealing well-defined structure in the aromatic region. The circular dichroism of NAD+ bound to glutamate dehydrogenase is strongly negative at 260 nm, 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. Glutarate and succinate, substrate analogues that are both inhibitors competitive with glutamate, do not significantly perturb the circular dichroic spectrum of the enzyme in the absence of NAD+. In the presence of NAD+, succinate (150 mM) decreases the negative circular dichroism corresponding to bound coenzyme, but does not affect the protein circular dichroism. Glutarate (150 mM) causes profound alterations of the circular dichroic spectra of the 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. The conformational change is discussed in relation to the allosteric mechanism of glutamate dehydrogenase.

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