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.

Inhibition of IMPDH by mycophenolic acid: dissection of forward and reverse pathways using capillary electrophoresis.

The objective of this work was to contribute to the understanding of mechanisms for IMPDH inhibition. We over-expressed hamster type II IMPDH in Escherichia coli, purified the protein to apparent homogeneity, and used capillary electrophoresis to quantify enzyme turnover events accompanying inhibition by mycophenolic acid (MPA). We dissected two convergent pathways leading to MPA-inhibition; a rapid "forward" pathway beginning with substrates and linked to enzyme catalysis, and a slower "reverse" pathway apparently not involving catalysis. MPA-inhibition occurred rapidly in the forward direction by interrupting the enzyme turnover cycle, after IMP and NAD+ binding, after hydride transfer, and after NADH release. Slow inhibition, without substrate turnover, was achieved by incubating free enzyme with excess XMP and MPA. We propose that mycophenolic acid inhibits IMPDH by trapping a transient covalent product of the hydride transfer reaction (IMPDH approximately XMP*) before a final hydrolysis step that precedes XMP and enzyme release in the forward reaction pathway. Understanding the ligand occupancy of the protein has also proven important for producing homogeneous, chemically defined complexes for structural studies. IMPDH samples inhibited by MPA in the forward and reverse pathways yielded similar, high-quality crystals that are currently undergoing X-ray diffraction analyses.

Enthalpy of hydrogen bond formation in a protein-ligand binding reaction.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding to FK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effort to probe the energetic contributions of single protein-ligand hydrogen bonds formed in the binding reactions. Changing tyrosine-82 to phenylalanine in FKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12 complexes with tacrolimus or rapamycin and leads to a large apparent enthalpic stabilization of binding in both H2O and D2O. High-resolution crystallographic analysis reveals that two water molecules bound to the tyrosine-82 hydroxyl group in unliganded FKBP-12 are displaced upon formation of the protein-ligand complexes. A thermodynamic analysis is presented that suggests that the removal of polar atoms from water contributes a highly unfavorable enthalpy change to the formation of C=O...HO hydrogen bonds as they occur in the processes of protein folding and ligand binding. Despite the less favorable enthalpy change, the entropic advantage of displacing two water molecules upon binding leads to a slightly more favorable free-energy change of binding in the reactions with wild-type FKBP-12.

Probing hydration contributions to the thermodynamics of ligand binding by proteins. Enthalpy and heat capacity changes of tacrolimus and rapamycin binding to FK506 binding protein in D2O and H2O.

The stabilities of native proteins and protein-ligand complexes result from differential interactions among numerous polar and nonpolar atoms within the proteins and ligands and of these atoms with water. Delineation of the various energetic contributions of the stabilities of proteins or protein-ligand complexes in aqueous solution, and an evaluation of their structural basis, requires a direct account of the changes, in the interactions of the protein with the solvent, that accompany the folding or binding reactions. Two largely nonpolar, structurally related macrolide ligands, tacrolimus (also known as FK506) and rapamycin, each bind with high affinity to a common site on a small FK506 binding protein (FKBP-12) and inhibit its peptidylprolyl cis-trans-isomerase activity. In an effort to elucidate the influence of water on the thermodynamics of their binding reactions, we have measured the enthalpies of tacrolimus and rapamycin binding to FKBP-12, in buffered solutions of H2O (at pH 7.0) or D2O (at pD 7.0), by high-precision titration calorimetry in the temperature range 5-30 degrees C. For both tacrolimus and rapamycin binding, a large enthalpic destabilization of binding is observed in D2O relative to H2O, in the temperature range examined. Additionally, large negative constant pressure heat capacity changes are observed for the binding of the ligands in both H2O and D2O. A thermodynamic analysis is presented to identify the structural determinants of the differences in the energetics of binding in light and heavy water. The analysis suggests that a chief contributor to the observed enthalpic destabilization is the differential hydration, of protein and ligand atoms, by light and heavy water.

Resonance Raman spectroscopy of chemically modified hemoglobins.

Hemoglobin obtained from out-dated human blood was stripped of 2,3-diphosphoglycerate and modified with the crosslinking agents glyoxalic acid, 1,2-cyclohexadione, or fumarate to stabilize the tetramer. The resulting hemoglobins, which show alterations in their oxygen transport capability, have been studied in their oxy, deoxy and fluoro-met forms using resonance Raman spectroscopy with Soret excitation. The resonance Raman spectra of oxy-hemoglobins cross linked with glyoxalic acid and 1,2-cyclohexadione show that these cross linking agents force the heme into a high spin structure. The resonance Raman spectra of the fluoro-met hemoglobins, however, indicate that the same cross linking agents force the heme into a lower spin structure. Absorption spectroscopy and molecular orbital considerations suggest that protein constraints at the sixth ligand of the heme can account for the change in spin state in the glyoxalic acid and 1,2-cyclohexadione cross linked hemoglobins.