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Recombinant Anthranilate Synthase From the Marine Hyperthermophile Archaeoglobus Fulgidus: Expression, Purification, Characterization and Stabilization of the Cooperatively-Regulated Heterotetrameric Enzyme

Published

Author(s)

W M. Byrnes, V L. Vilker

Abstract

Thermostable anthranilate synthase from the marine sulfate-reducing hyperthermophile Archaeoglobus fulgidus has been expressed in Escherichia coli, purified, and characterized. The functional enzyme is an α2β2 heterotetrameric complex of molecular weight 150 18 kDa; it is composed of two TrpE (50 kDa) and two TrpG (18 kDa) subunits. Glycerol (25%) and potassium chloride (2 M) stabilized the recombinant enzyme 56-fold against thermal inactivation. In the presence of these extrinsic stabilizing factors, the enzyme was highly thermostable, exhibiting a half-life of thermal inactivation of about one hour at 85 C. The kinetic and catalytic properties of the enzyme remained essentially unchanged in the presence versus the absence of potassium chloride and glycerol, with two exceptions: the Km(chorismate), which increased 7-fold, and the pH optimum, which decreased 0.8 pH unit. The enzyme was strongly inhibited by tryptophan (Ki = 0.7 M), and exhibited a noncooperative response to chorismate in the presence of tryptophan when glycerol and potassium chloride were absent, but a highly cooperative response in the presence of these two solutes. An explanation is presented for how these extrinsic factors (glycerol and salt) might stabilize the recombinant oligomeric enzyme and promote cooperative allosteric interactions among its subunits.
Citation
Biochemistry

Keywords

allosteric, anthranilate synthase, glycerol, hyperthermophile, salt, thermostable

Citation

Byrnes, W. and Vilker, V. (2008), Recombinant Anthranilate Synthase From the Marine Hyperthermophile Archaeoglobus Fulgidus: Expression, Purification, Characterization and Stabilization of the Cooperatively-Regulated Heterotetrameric Enzyme, Biochemistry (Accessed June 25, 2024)

Issues

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Created October 16, 2008