A Six-State Binding Model Gives Rise To Dynamic Activity And Energy Landscapes In Yeast Chorismate Mutase

The biosynthesis of amino acids is energetically costly. As such, pathway end-products often allosterically regulate the involved enzymes in order to ensure appropriate levels of amino acids. Many of the enzymes involved in amino acid biosynthesis are multimeric, which allows for cooperative interactions between allosteric sites to help fine-tune enzyme activities. Saccharomyces cerevisiae chorismate mutase (ScCM) is an excellent model system for studying allosteric regulation because it is a homodimeric enzyme capable of binding both an inhibitor, Tyr, and an activator, Trp, to the same two equivalent allosteric sites. Through the complementary techniques of isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy, we have demonstrated that under cellular concentrations of Tyr and Trp, ScCM fluctuates between states in which one Tyr or Trp is bound, states in which both allosteric sites are occupied by Tyr or by Trp, and a mixed state in which Tyr is bound to one allosteric site and Trp is bound to the other. The interplay between these states and their effect on the overall chorismate mutase activity was then investigated through specific activity assays, providing estimates of the relative activities of each state and predictions of relative enzyme activities under in vivo conditions. The ability of ScCM to bind only a single Tyr or Trp is predicated on the negative cooperativity between the allosteric binding sites, which ITC indicated is due to the entropic cost of binding. To better understand this entropic cost, we measured methyl axis order parameters by NMR, which provide an indication of changes to protein conformational entropy upon binding, and conducted multi-temperature ITC measurements to determine heat capacity change. These results provide new insights into the role of water in governing allosteric regulation.