Urea-induced inactivation and denaturation of clostridial glutamate dehydrogenase: the absence of stable dimeric or trimeric intermediates

Abstract

Urea-induced effects in clostridial glutamate dehydrogenase (GDH, EC 1.4.1.2) were studied by spectrophotometry, circular dichroism, FPLC, affinity chromatography and PAGE. Denaturation of enzyme occurred over a narrow range of urea concentrations (2.5-3.5 M), accompanied by inactivation of enzyme with a similar rate constant. The contribution of instantaneous inhibition by urea was also ascertained. FPLC studies of urea-treated GDH gave no evidence for dissociated oligomeric fragments of the hexamer in the presence of subdenaturing concentrations of urea. Likewise a mixture of fully 5,5'-dithiobis-(2-nitrobenzoic acid)-modified GDH hexamers and unmodified enzyme in 2 M urea failed to give rise to hybrid molecules. Exposure of unmodified GDH to high concentrations of urea led to the dissociation of hexamers to denatured monomers followed by association to form non-specific high-M(r) aggregates. This conclusion was confirmed by native gradient PAGE experiments. Various specific ligands stabilized the enzyme against urea-induced inactivation, succinate and 2-oxoglutarate being particularly effective. This protection of the native state was enhanced in ternary complexes, and the complex most resistant to urea-induced inactivation was the productive ternary complex GDH-NADH-2-oxoglutarate. Native gradient PAGE experiments indicate that these protecting ligands preserve the native hexameric structure of GDH.

Re-activation of Clostridium symbiosum glutamate dehydrogenase from subunits denatured by urea

“…Most GDHs are hexamers composed of identical subunits arranged as a dimer of trimers [1][2][3][4][5][6][7][8][9][10][11][12]. A number of studies of the unfolding of both soluble and immobilized GDHs in the presence of denaturing agents such as guanidinium chloride (GdmCl) and urea have been made [13][14][15][16][17][18][19][20][21][22][23][24][25][26]. For the clostridial enzyme, for which a high-resolution structure is available [10], it was concluded that, at concentrations of GdmHCl insufficient to cause denaturation, the hexamer dissociates into inactive trimers without any gross structural changes that could be detected by fluorescence or CD [20].…”

“…NAD + alone was also effective but 2-oxoglutarate was not, in striking contrast with the opposite pattern for protection of this enzyme against unfolding in urea [Aghajanian, Martin and Engel (1995) Biochem. J.…”

Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity

“…To unfold and separate the subunits [28], the two proteins, each at 1.5 mg\ ml protein concentration in 0.1 M potassium phosphate buffer (pH 7.0), were incubated separately at 25 mC in 4.5 M urea for 60 min (triple mutant) or 4 M urea for 30 min (C320S). The two denatured enzymes (residual activities 1 %) were mixed at a 1 : 5 (triple mutant :C320S) volume ratio, diluted 25-fold into 0.1 M potassium phosphate buffer (pH 7.0) containing 2 mM NAD + , and incubated at 25 mC for 36 h for refolding and reactivation as described previously [16].…”

Kinetic analysis of urea-inactivation of β-galactosidase in the presence of galactose

“…In other words, as viewed from the energy landscape theory or the folding funnel concept [3 -7], the native state of a protein is that minimally frustrated heteropolymer with a rugged funnel-like landscape biased towards the native structure [8,9]. The particular conformation taken by the protein influences its biological activity [10,11] and this has influenced the comparison though by limited authors, of the conformation and activity changes during the course of enzyme denaturation [12].…”