Research on allosteric threonine deaminase from Escherichia coli will reveal protein interactions that regulate enzyme activity. Cloning and high-level expression will aid in purifying wild-type and mutant forms of threonine deaminase for their biochemical characterization. Analysis of the protein concentration dependence of enzyme kinetic parameters will elucidate a model for the transition between low and high activity conformations. Identifying malfunctioning variants by classical genetics will reveal structural domains involved in catalysis and regulation, and their effect on the folding of polypeptides will be evaluated by x-ray crystallography. Sedimentation equilibrium studies of the tetramer-dimer interconversion of threonine deaminase will permit quantitative determination of changes in subunit interaction energy. Direct ligand binding measurements by equilibrium dialysis, coupled with the energetics of dimer- tetramer assembly, will reveal the thermodynamic basis for cooperativity. The cofactor pyridoxal 5'-phosphate, a chromophore in the active site, can reveal local changes promoted by effector binding that alter catalysis. Quantitative analysis of absorption spectra in terms of sums of lognormal curves will establish a free energy profile for the enzyme to assess the effect of heterotropic ligands on each step of the reaction pathway. Presteady-state kinetic studies will correlate the kinetic and thermodynamic effects of inhibitor binding.
