Delineation of Alternative Conformational States in Escherichia coli Peptide Deformylase via Thermodynamic Studies for the Binding of Actinonin

We investigated the binding of a naturally occurring antibiotic, actinonin, to the Ni2+-reconstituted recombinant form of Escherichia coli peptide deformylase (PDFEc) via isothermal titration microcalorimetry. The binding data conformed to both exothermic and endothermic phases with magnitudes of Delta G degrees, Delta H degrees, and T Delta S degrees being equal to -12, -2.7, and 9.3 kcal/mol and -8.7, 3.9, and 12.6 kcal/mol, respectively. Evidently, although both phases are dominated by favorable entropic changes, the exothermic phase has about 6.7 kcal/mol enthalpic advantage over the endothermic phase. We observed that the removal of bound Ni2+ from PDFEc abolished the exothermic phase without affecting the endothermic phase, but it was regained upon addition of Zn2+. In conjunction with metal analysis data, we propose that the recombinant form of PDFEc is expressed in two stable conformational states that yield markedly distinct ITC profiles (i.e., exothermic versus endothermic) upon interaction with actinonin. The existence of two conformational states of PDFE,, is further supported by the observation of two distinct and independent transitions during the thermal unfolding of the enzyme. In addition, the thermodynamic data reveal that the formation of the PDFEc-actinonin complex results in the transfer of one H+ from the enzyme phase to the bulk solvent at pH 6.3. Both exothermic and endothermic phases produce highly negative Delta C-p degrees values, but there is no apparent enthalpy-entropy compensation effect upon formation of the PDFEc-actinonin complex. In view of the known structural features of the enzyme, arguments are presented that the alternative conformational states of PDFEc are modulated by the metal ligation at the enzyme site.