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,
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.)