Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S

Abstract

In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both glutamate and NAD 1 but is catalytically inactive. The mutant proteins were denatured separately in 4 m urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mm NAD 1 , 1 m fluoride and artificial chaperones (4 mm polyoxyethylene 10 lauryl ether and 1.6 mm b-cyclodextrin). Under these conditions < 50% refolding was achieved for both mutant proteins separately. The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion-exchange chromatography. Ellman's reagent, 5,5H -dithiobis (2-nitrobenzoic acid) (DTNB), which binds close to the NAD 1 binding site, thus abolishing coenzyme binding in the wild-type enzyme, also reacts with D165S but has no effect on C320S. Modification by DTNB was coupled with dye-ligand affinity chromatography on a Procion Red HE-3B column in order to separate the hybrid mixture into fractions of defined composition. An optimized procedure based on salt gradient elution was developed. DTNB-modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis± Menten kinetics when the NAD 1 concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that nonlinear' behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites. When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h 3.0) compared with native C320S with six active subunits (h 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely. Catalytically silent subunits can thus still contribute to cooperativity.Keywords: allosteric interaction; glutamate dehydrogenase; subunit hybrids.Glutamate dehydrogenases (GDHs; EC 1.4.1.2±4) have been the object of much attention following the discovery that GDHs from many sources display complex regulatory behaviour and striking departure from classical Michaelis± Menten kinetics [1±4]. In the first report offering a possible mechanism of cooperativity, Monod et al. (1963) [1] cited bovine liver GDH as an example of an allosteric enzyme. However, the sheer complexity of this enzyme, coupled with the lack of a three-dimensional structure [5] (finally solved in 1999), made it difficult to unravel the basis of the allosteric behaviour. The homohexameric GDH from Clostridium symbiosum, which lacks the 48 amino acid insertion suspected of c...

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.…”

Homotropic allosteric control in clostridial glutamate dehydrogenase: Different mechanisms for glutamate and NAD+?

“…Mutational studies have revealed that each effect can persist in the absence of the other when binding sites are disabled (e.g. [16,17]), but this does not unambiguously indicate whether the two effects rely on septe structural machinery. Recently, however, pursuit of Trp residues thought to be sensing the pH/glutamate‐dependent conformational change [14], has highlighted two, W64 and W449, which appear to provide six pairwise interactions across the trimer–trimer interface [18].…”

Effect of selected compounds on the activity of glutamate dehydrogenase from triticale roots

“…Among the applied compounds, it was DTNB that proved the most efficient inhibitor to NADH-GDH isolated from bean leaves. Recently Hayden and Engel (2001) have shown an important role of-SH groups in coenzyme binding. Additionally, a review of relevant studies (Turano 1997) led to the conclusion that there exist two different binding sites for the substrate (2-oxoglutarate and L-glutamate) fixation into the enzyme molecule.…”