Specificity of coenzyme analogues and fragments in promoting or impeding the refolding of clostridial glutamate dehydrogenase

Abstract

NAD+ facilitates high-yield reactivation of clostridial glutamate dehydrogenase (GDH) after unfolding in urea. The specificity of this effect has been explored by using analogues and fragments of NAD+. The adenine portion, unlike the nicotinamide portion, is important for reactivation. Alteration in the nicotinamide portion, in acetylpyridine adenine dinucleotide, has little effect, whereas loss of the 6-NH2 substitution on the adenine ring, in 6-deamino NAD, diminishes the effectiveness of the nucleotide in promoting refolding. Also ADP-ribose, lacking nicotinamide, promotes reactivation whereas NMN-phosphoribose, lacking the adenine, does not. Of the smaller fragments, those containing an adenosine moiety, and especially those with one or more phosphate groups, impede the refolding ability of NAD+, and are able to bind to the folding intermediate though unable to facilitate refolding. These results are interpreted in terms of the known 3D structure for clostridial glutamate dehydrogenase. It is assumed that the refolding intermediate has a more or less fully formed NAD+-binding domain but a partially disordered substrate-binding domain and linking region. Binding of NAD+ or ADP-ribose appears to impose new structural constraints that result in completion of the correct folding of the second domain, allowing association of enzyme molecules to form the native hexamer.

Apparent negative co-operativity and substrate inhibition in overexpressed glutamate dehydrogenase from Escherichia coli

“…Our laboratory has been carrying out a study of structurefunction relationships in GDH from a variety of organisms (e.g. Syed et al, 1991;Basso et al, 1995;Rice et al, 1996;Aghajanian et al, 1999Aghajanian et al, , 2003Hayden & Engel, 2001;Hayden et al, 2002;Carrigan et al, 2005), including the basis of coenzyme specificity. On focusing attention to the NADP 1 -dependent EcGDH, we became aware of the lack of reliable baseline data.…”