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Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli.

Publication ,  Journal Article
Chinchilla, D; Schwarz, FP; Eisenstein, E
Published in: The Journal of biological chemistry
September 1998

Shifts in the sigmoidal kinetics of allosteric threonine deaminase promoted by isoleucine and valine binding control branched chain amino acid biosynthesis in Escherichia coli. A highly conserved alpha-helix in the C-terminal regulatory domain of the tetrameric enzyme was previously implicated in effector binding and feedback inhibition. Double (447, 451) and triple (447, 451, 454) alanine replacements for the conserved amino acids leucine 447, leucine 451, and leucine 454 in this region yield enzyme variants that show increased sigmoidality in steady-state kinetics, and which are less sensitive to the allosteric modifiers isoleucine and valine. Equilibrium binding studies using fluorescence, enzyme kinetic, and calorimetric approaches indicate that the enzyme variants possess reduced affinity for isoleucine and valine, and suggest that heterotropic ligands can bind to the same site to promote their different effects. The increase in sigmoidal kinetics for the mutants relative to wild-type threonine deaminase may be attributable to the elimination of L-threonine binding to the effector sites, which activates the wild-type enzyme. Enzyme kinetic data and isotherms for active site ligand binding to the mutants can be analyzed in terms of a simple two-state model to yield values for allosteric parameters that are consistent with previous estimates based on an expanded two-state model for homotropic cooperativity for threonine deaminase.

Duke Scholars

Published In

The Journal of biological chemistry

ISSN

0021-9258

Publication Date

September 1998

Volume

273

Issue

36

Start / End Page

23219 / 23224

Location

united states

Related Subject Headings

  • Biochemistry & Molecular Biology
  • 11 Medical and Health Sciences
  • 06 Biological Sciences
  • 03 Chemical Sciences
 

Citation

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Chinchilla, D., Schwarz, F. P., & Eisenstein, E. (1998). Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli. The Journal of Biological Chemistry, 273(36), 23219–23224.
Chinchilla, D., F. P. Schwarz, and E. Eisenstein. “Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli.The Journal of Biological Chemistry 273, no. 36 (September 1998): 23219–24.
Chinchilla D, Schwarz FP, Eisenstein E. Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli. The Journal of biological chemistry. 1998 Sep;273(36):23219–24.
Chinchilla, D., et al. “Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli.The Journal of Biological Chemistry, vol. 273, no. 36, Sept. 1998, pp. 23219–24.
Chinchilla D, Schwarz FP, Eisenstein E. Amino acid substitutions in the C-terminal regulatory domain disrupt allosteric effector binding to biosynthetic threonine deaminase from Escherichia coli. The Journal of biological chemistry. 1998 Sep;273(36):23219–23224.

Published In

The Journal of biological chemistry

ISSN

0021-9258

Publication Date

September 1998

Volume

273

Issue

36

Start / End Page

23219 / 23224

Location

united states

Related Subject Headings

  • Biochemistry & Molecular Biology
  • 11 Medical and Health Sciences
  • 06 Biological Sciences
  • 03 Chemical Sciences