A COMBINATION OF GENETIC AND BIOPHYSICAL MEASUREMENTS REVEALS CONTROL FEATURES OF THE ALLOSTERIC THREONINE DEAMINASE. Diana Chinchilla & Edward Eisenstein. Center for Advanced Research in Biotechnology, Rockville, MD. (CARB, Room 217, 9600 Gudelsky Drive, Rockville, MD 20850, 301-738-6152, email: dianach@indigo3.carb.nist.gov)

Branched-chain amino acid biosynthesis in plants and microorganisms is controlled by feedback regulation of allosteric threonine deaminase (TD). Biophysical measurements on genetic variants of TD are being used to resolve a molecular model for control of cooperative substrate binding and catalysis. Capitalizing on the crystal structure of TD, dimeric variants have been engineered that display sigmoidal steady-state kinetics, and cooperative isoleucine and valine binding. Interestingly, analyses of binding isotherms performed as a function of protein concentration and sedimentation studies to measure the dimer-tetramer equilibrium show unequivocally that active site ligands stabilize the tetramer, and indicate that the cooperative free energy for substrates is negative. In contrast, TD variants with amino acid substitutions in a strongly conserved helix in the regulatory domain exhibit markedly altered allosteric properties, and suggest a location for heterotropic effector binding. Although the mutants display cooperative enzyme kinetics with velocities comparable to wild-type TD, they are insensitive to feedback modifiers. Both fluorescence and calorimetric measurements indicate that the mutants exhibit a 15- to 100-fold reduction in their affinity for isoleucine, and valine binding is undetectable. The allosteric parameters for the mutants due solely to homotropic cooperativity are in excellent agreement with those predicted from analyses of wild- type TD. (Supported by NSF grant MCB 93-04940.)