Reversible pH-dependent inactivation of glutamate dehydrogenase from Clostridium symbiosum

1986 ◽  
Vol 14 (1) ◽  
pp. 157-157 ◽  
Author(s):  
SHABIH-E-HASSNAIN SYED ◽  
PAUL C. ENGEL
Keyword(s):  
Glutamate Dehydrogenase ◽  
Dependent Inactivation ◽  
Clostridium Symbiosum ◽  
Ph Dependent

scholarly journals A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum

1990 ◽  
Vol 271 (2) ◽  
pp. 351-355 ◽  
Author(s):  
S E H Syed ◽  
P C Engel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Time Course ◽  
Enzyme Concentration ◽  
Complete Inactivation ◽  
Activation Rate ◽  
Clostridium Symbiosum ◽  
Activity Form ◽  
Ph Dependent ◽  
Time Courses ◽  
Low Ionic Strength

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.

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.

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
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