9723086 Eisenstein This research aims to forge a quantitative mechanism for the allosteric regulation of substrate binding and catalysis in a family of pyridoxal phosphate-dependent threonine deaminases. The approach consists of analyzing complementary information from structural, genetic, thermodynamic and kinetic studies to yield more accurate insights into the fundamental interactions between subunits that give rise to cooperative ligand binding and feedback regulation. Refining the crystal structure to 2.3 angstroms in the presence of feedback modifiers and substrate analogs will shed light on the structural basis for the allosteric transition. Variations in the energetics of dimer-tetramer assembly for mutant variants in combination with ligand binding studies will reveal changes in subunit interaction energies that are linked to cooperative ligand binding. Additionally, sigmoidal isoleucine and valine binding to fully active, dimeric variants will reveal the cooperative free energies associated with heterotropic effector binding. Heterodimeric hybrid enzymes containing different arrangements of functional catalytic and regulatory sites will be used to assess whether heterotropic effectors promote global or only local regulation. Substrate and solvent isotope effects in steady state and pre-steady state kinetics will be used to establish the effects of isoleucine and valine on the rate-limiting step(s) in catalysis. This research aims to provide complementary information from diverse experimental strategies to develop a molecular level understanding of the regulation of catalysis in the threonine deaminase family of pyridoxal phosphate-requiring allosteric enzymes. The biological activity of allosteric proteins is modulated by small molecules called effectors. Binding of effector molecules results in the propagation of widespread structural changes that lead to biologically important changes in function. This tenet provides a central paradigm in biological chemistry, which ul timately will guide the design of control features into novel proteins.
