Amino Acid Substitutions in the C-Terminal Regulatory Domain Disrupt Allosteric Effector Binding to Biosynthetic Threonine Deaminase from Escherichia Coli

Published

June 1, 1999

Author(s)

D Chinchilla, Frederick P. Schwarz, Edward Eisenstein

Abstract

Shifts in the sigmoidal kinetics of allosteric threonine deaminase promoted by isoleucine and valine binding control branched chain amino acid biosynthesis in Each-erichia coli. A highly conserved α-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 wildd type threonine deaminase may be attributable to the elimination of L-threonin 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 that are consistent with previous estimates based on an expanded two-state model for homotropic cooperativity for threonine deaminase.

Citation

Journal of Biological Chemistry

Volume

273

Pub Type

Journals

Keywords

allostery, cooperativity, feedback modifiers, pyridoxal phosphate, regulation

Citation

Chinchilla, D. , Schwarz, F. and Eisenstein, E. (1999), Amino Acid Substitutions in the C-Terminal Regulatory Domain Disrupt Allosteric Effector Binding to Biosynthetic Threonine Deaminase from Escherichia Coli, Journal of Biological Chemistry (Accessed April 26, 2024)