Allosteric properties of inosine monophosphate dehydrogenase revealed through the thermodynamics of binding of inosine 5'-monophosphate and mycophenolic acid. Temperature dependent heat capacity of binding as a signature of ligand-coupled conformational equilibria.
The thermodynamic properties of binding of the substrate, inosine monophosphate (IMP), and the uncompetitive inhibitor, mycophenolic acid, to inosine monophosphate dehydrogenase (IMPDH) were measured. Specifically, the free energy, enthalpy, entropy, and heat capacity changes were 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 negatively cooperative fashion and that mycophenolic acid binding is critically dependent on the presence of IMP. Moreover, the binding of IMP is 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 nature of the heat capacity function for a binding reaction and its temperature dependence, is used to infer the existence of an equilibrium mixture of at least two structural forms of apo-IMPDH. The equilibrium is perturbed in the presence of IMP and mycophenolic acid, suggesting a mechanism for the ligand-linked conformational changes. An allosteric model, incorporating subunit-subunit interactions nested within a concerted conformational change involving the entire tetrameric macromolecule, is proposed to account for the observed binding behavior. The implications of these findings for the design of novel "allosteric-effector" inhibitors of IMPDH, to be used for the purpose of immunosuppression, are discussed.
Bruzzese
F J
FJ
Vertex Pharmaceuticals Incorporated, 40 Allston Street, Cambridge, Massachusetts 02139, USA.
Connelly
P R
PR
D000494:Allosteric Regulation; D000818:Animals; D002151:Calorimetry; D006224:Cricetinae; D015195:Drug Design; D007168:IMP Dehydrogenase / Q000378:metabolism*; D007291:Inosine Monophosphate / Q000378:metabolism* / Q000494:pharmacology; D008024:Ligands; D009173:Mycophenolic Acid / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics
D008024:Ligands; D007291:Inosine Monophosphate; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid