The energy and enzyme activity landscapes of yeast chorismate mutase under cellular concentrations of allosteric effectors

In solution, proteins fluctuate among many conformational substates, with their relative free energies determining sub-state populations and energy barriers determining conformational exchange kinetics. It has been suggested that members of the conformational ensemble may be responsible for different protein functions, although it is generally difficult to test such a proposal in most systems. A model protein for deciphering individual substate contributions is the ho-modimeric Saccharomyces cerevisiae chorismate mutase (ScCM) enzyme, which is positively and negatively regulated by tryptophan and tyrosine, respectively. Previous X-ray crystallography structures revealed two equivalent allosteric binding pockets that can be occupied by either tryptophan or tyrosine. We proposed that under cellular conditions there are six potential states of ScCM: no allosteric effector bound, a single tyrosine bound, a single tryptophan bound, two tyro-sines bound, two tryptophans bound, and a mixed bound state in which tyrosine and tryptophan occupy different allo-steric sites. We used isothermal titration calorimetry and solution state nuclear magnetic resonance spectroscopy to con-firm the existence of all six states and construct the complete six-state equilibrium binding profile. We were also able to assign enzyme activities to each state, which allowed us to derive the enzyme activity landscape across the range of cellu-lar concentrations of tyrosine and tryptophan. Surprisingly, the mixed bound state had the highest enzyme activity, which suggested that the shikimate pathway is shunted towards tyrosine production under most conditions.