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.
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December 2001
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Research Article|
December 10 2001
Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity
Arun GOYAL;
Arun GOYAL
1
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland
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Xing-Guo WANG;
Xing-Guo WANG
2
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland
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Paul C. ENGEL
Paul C. ENGEL
3
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland
3To whom correspondence should be addressed (e-mail paul.engel@ucd.ie).
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Publisher: Portland Press Ltd
Received:
June 28 2001
Revision Received:
September 24 2001
Accepted:
October 03 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 360 (3): 651–656.
Article history
Received:
June 28 2001
Revision Received:
September 24 2001
Accepted:
October 03 2001
Citation
Arun GOYAL, Xing-Guo WANG, Paul C. ENGEL; Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity. Biochem J 15 December 2001; 360 (3): 651–656. doi: https://doi.org/10.1042/bj3600651
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