An important consideration in the design of enzyme inhibitors is their solubilities in aqueous solution. In fact, most of the aspartic proteinases inhibitors and substrates currently available show very poor solubility in water. As a consequence, many of the structural , kinetic and thermodynamic studies involving such molecules can only be performed in water/organic solvent mixtures. Dimethylsulfoxide (DMSO) has been extensively used for such purpose. We have studied the effect of dimethylsulfoxide on the energetics of the inhibitor/enzyme interaction. In the binding of pepstatin A to endothiapepsin, the enthalpy changes decrease slightly but monotonically with the addition of increasing amounts of DMSO from -7.4 kcal/mol in pure sodium formate buffer to -8.4 kcal/mol in the same buffer containing 10% DMSO at 25 oC and pH 3.1. Nevertheless, the heat capacity change upon binding (-310 cal/K mol) remains unaffected within the experimental error by the presence of DMSO. Similar results were obtained for the binding of pepstatin A to porcine pepsin. At the highest DMSO content tested (10%), no deleterious effect on the ability of pepstatin A to inhibit the enzyme was detected. The inhibition constants at 25 oC and pH 3.1 were 3 nM in pure buffer and 2 nM in 10% (v/v) DMSO. Therefore, the free energy change upon binding is largely independent of the DMSO concentration within the concentration range studied, indicating that the decrease in enthalpy change is entropically compensated. Once the role of DMSO on the energetics of the enzyme inhibition has been clarified, we have thermodynamically characterized the binding reaction between endothiapepsin and several pepstatin derivatives by means of isothermal titration calorimetry and structure based thermodynamic analysis
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