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

Abstract

In a study of the re-activation of urea-denatured clostridial glutamate dehydrogenase (GDH) the maximum re-activation achieved without any added ligands was about 6 %, but with NAD + and 2-oxoglutarate in combination about 70 %. 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. 311, [905][906][907][908][909][910]. The extent of re-activation was not increased by raising the incubation temperature to 37 mC and was independent of the time of enzyme denaturation. CD and fluorimetric studies showed that dilution of denatured enzyme into potassium phosphate buffer led to rapid (half-time 3-5 s) formation of ' structured ' intermediates with secondary structure similar to that of native enzyme. These intermediate molecules were inactive, behaved as monomers on a size-exclusion column,

Overexpression in a non-native halophilic host and biotechnological potential of NAD+-dependent glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014

“…NAD ? -dependent glutamate dehydrogenase from Clostridium symbiosum (Aghajanian and Engel 1997), the stabilizing effect of glutamate and 2-oxoglutarate on GDHX was also studied under suboptimal salt concentrations. The residual activity was measured in duplicate samples.…”

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]. Controls were run for both the mutants to check the effect of incubation without urea treatment, and later of mixing and diluting the two mutants.…”

“…A route to a more systematic exploration of inter-subunit communication has been provided by the successful refolding of the enzyme [16], allowing the construction of hybrid hexamers. Defined inter-subunit hybrids have been made by combining chemical modification and site-directed mutagenesis [17][18][19][20].…”

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

“…The inter-subunit contacts in the GDHs of prokaryotes at least are formed exclusively between residues of the latter domain in each subunit~Baker et al, 1992a!. For reasons that are not clear, GDHs have been remarkably resistant to reversible unfolding~Eisenberg et al, 1976;Sugrobova et al, 1979;Müller & Jaenicke, 1980;West & Price, 1988!. Recently, however, some bacterial GDHs have been successfully re- folded~Aghajanian & Engel, 1994, 1997aGuagliardi et al, 1994;Consalvi et al, 1995Consalvi et al, , 1996!. The GDH of Clostridium symbiosum, for which the most detailed structural information is availablẽ Baker et al, 1992a!, has been the subject of a detailed study of unfolding by urea~Aghajanian et al, 1995!…”

“…A particularly interesting aspect of this has been the contrasting effects of coenzymes and dicarboxylate ligands on the two processes. In the case of unfolding, the coenzymes are essentially without effect, whereas 2-oxoglutarate~50 mM!, for instance, shifts the concentration of urea that gives 50% inactivation in 1 h from 2.4 to 3.9 M~Aghajanian et al, 1995!. The opposite pattern is seen in relation to the process of refold-ing~Aghajanian & Engel, 1994, 1997a!. Dilution of urea-denatured clostridial GDH into potassium phosphate buffer results in recovery of the enzyme's secondary structure, although most of the structured molecules remain as inactive monomers and are unable to reform into active hexamers~Aghajanian & Engel, 1997a!.…”