ALLOSTERIC PROPERTIES OF INOSINE MONOPHOSPHATE DEHYDROGENASE REVEALEDTHROUGH THE THERMODYNAMICS OF BINDING OF INOSINE 5'-MONOPHOSPHATE ANDMYCOPHENOLIC-ACID - TEMPERATURE-DEPENDENT HEAT-CAPACITY OF BINDING ASA SIGNATURE OF LIGAND-COUPLED CONFORMATIONAL EQUILIBRIA

The thermodynamic properties of binding of the substrate, inosine mono phosphate (IMP), and the uncompetitive inhibitor, mycophenolic acid, t o inosine monophosphate dehydrogenase (IMPDH) were measured. Specifica lly, the free energy, enthalpy, entropy, and heat capacity changes wer e determined for each ligation state of the tetrameric enzyme, over a temperature range from 2.5 to 37 degrees C by high-precision titration microcalorimetry. It was discovered that IMP binds to IMPDH in a nega tively cooperative fashion and that mycophenolic acid binding is criti cally dependent on the presence of IMP. Moreover, the binding of IMP i s entropically driven at low temperatures and enthalpically driven at high temperatures, with an unusually large, temperature dependent heat capacity change. A thermodynamic argument, based on the general natur e of the heat capacity function for a binding reaction and its tempera ture dependence, is used to infer the existence of an equilibrium mixt ure of at least two structural forms of apo-IMPDH. The equilibrium is perturbed in the presence of IMP and mycophenolic acid, suggesting a m echanism for the ligand-linked conformational changes. An allosteric m odel, incorporating subunit-subunit interactions nested within a conce rted conformational change involving the entire tetrameric macromolecu le, is proposed to account for the observed binding behavior. The impl ications of these findings for the design of novel ''allosteric-effect or'' inhibitors of IMPDH, to be used for the purpose of immunosuppress ion, are discussed.