THERMODYNAMICS OF A TRANSITION-STATE ANALOG INHIBITOR BINDING TO ESCHERICHIA-COLI CHORISMATE MUTASE - PROBING THE CHARGE-STATE OF AN ACTIVE-SITE RESIDUE AND ITS ROLE IN INHIBITOR BINDING AND CATALYSIS

Electrostatic interactions play important roles in the catalysis of ch orismate to prephenate by chorismate mutase. Mutation of Gln88 to glut amate in the monofunctional chorismate mutase from Escherichia coli re sults in an enzyme with a pH profile of activity significantly differe nt from that of the wild type protein. To investigate whether the muta tion alters the substrate binding process or the catalysis, we have di rectly determined the thermodynamic parameters of a transition state a nalogue inhibitor binding to the wild-type chorismate mutase and its Q 88E mutant using isothermal titration calorimetry. The results demonst rate that solvent reorganization and hydrophobic interactions contribu te the predominant free energy to inhibitor binding. The charge state of Glu88 in the Q88E mutant was experimentally determined and was show n to be protonated at pH 4.5 and ionized at pH 7.8, consistent with ea rlier hypotheses. Most surprisingly, inhibitor binding energetics do n ot exhibit significant pH dependency for both enzymes. Our findings in dicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.