Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces

1997 ◽  
Vol 25 (5-6) ◽  
pp. 417-422 ◽  
Author(s):  
Jonathan L. E. Dean ◽  
H. Cölfen ◽  
Stephen E. Harding ◽  
David W. Rice ◽  
P. C. Engel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Quaternary Structure ◽  
Single Mutation ◽  
Clostridium Symbiosum

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

2001 ◽  
Vol 360 (3) ◽  
pp. 651-656 ◽  
Author(s):  
Arun GOYAL ◽  
Xing-Guo WANG ◽  
Paul C. ENGEL
Keyword(s):  
Amino Acid ◽  
Glutamate Dehydrogenase ◽  
Cysteine Residue ◽  
The Other ◽  
Wild Type Enzyme ◽  
Triple Mutant ◽  
Subunit Stoichiometry ◽  
Clostridium Symbiosum ◽  
Mutant Forms ◽  
Fold Activation

Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD+–agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40mM glutamate and 1mM NAD+ at pH8.8, or K89L/A163G/S380A with 70mM norleucine and 1mM NAD+ at pH8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC50 = 70mM, h = 2.4), and glutarate gave approx. 30-fold activation (EC50 = 35mM, h = 2.3). For the triple mutant, corresponding values were 80mM and 2.2 for succinate, and 75mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The ‘foreign’ subunit evidently impedes inter-subunit communication to some extent.

scholarly journals Crystal structure of a chimaeric bacterial glutamate dehydrogenase

2016 ◽  
Vol 72 (6) ◽  
pp. 462-466 ◽  
Author(s):  
Tânia Oliveira ◽  
Michael A. Sharkey ◽  
Paul C. Engel ◽  
Amir R. Khan
Keyword(s):  
Crystal Structure ◽  
Glutamate Dehydrogenase ◽  
Rigid Bodies ◽  
Nucleotide Binding Domain ◽  
Oxidative Deamination ◽  
E Coli ◽  
Cofactor Binding ◽  
Signature Sequences ◽  
Clostridium Symbiosum ◽  
Domain I

Glutamate dehydrogenases (EC 1.4.1.2–4) catalyse the oxidative deamination of L-glutamate to α-ketoglutarate using NAD(P)+as a cofactor. The bacterial enzymes are hexameric, arranged with 32 symmetry, and each polypeptide consists of an N-terminal substrate-binding segment (domain I) followed by a C-terminal cofactor-binding segment (domain II). The catalytic reaction takes place in the cleft formed at the junction of the two domains. Distinct signature sequences in the nucleotide-binding domain have been linked to the binding of NAD+versusNADP+, but they are not unambiguous predictors of cofactor preference. In the absence of substrate, the two domains move apart as rigid bodies, as shown by the apo structure of glutamate dehydrogenase fromClostridium symbiosum. Here, the crystal structure of a chimaeric clostridial/Escherichia colienzyme has been determined in the apo state. The enzyme is fully functional and reveals possible determinants of interdomain flexibility at a hinge region following the pivot helix. The enzyme retains the preference for NADP+cofactor from the parentE. colidomain II, although there are subtle differences in catalytic activity.

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

1997 ◽  
Vol 326 (3) ◽  
pp. 649-655 ◽  
Author(s):  
Suren AGHAJANIAN ◽  
Paul C. ENGEL
Keyword(s):  
Glutamate Dehydrogenase ◽  
Time Course ◽  
Incubation Temperature ◽  
Specific Activity ◽  
Potassium Phosphate ◽  
Enzyme Concentration ◽  
Size Exclusion ◽  
Native Enzyme ◽  
Opposite Pattern ◽  
Clostridium Symbiosum

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, 905–910]. The extent of re-activation was not increased by raising the incubation temperature to 37 °C 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, and were unable to associate to give the native hexameric structure. Addition of NAD+ facilitated isomerization of these ‘structured’ monomers into a form(s) capable of re-activation. A side effect in the refolding process was non-specific aggregation, depending on final enzyme concentration. The hexamer fraction from re-activated samples, however, showed the same specific activity as native enzyme. The portion of the enzyme that is not lost through aggregation thus appears to regain the native structure fully. Detailed time-course studies showed that re-activation follows second-order kinetics, suggesting that formation of a dimer may be the rate-limiting step. The possible mechanism for the unfolding and refolding processes of clostridial GDH and effects of coenzyme and substrate on these are discussed in relation to the known crystal structure.

Sign in / Sign up

Export Citation Format

Share Document