Thermodynamic characterization of ligand-induced conformational changes inUDP-N-acetylglucosamine enolpyruvyl transferase

The binding of UDP-N-acetylglucosan-Line (UDPNAG) to the enzyme UDP-N-acety lglucosamine enolpyruvyl transferase (MurA) was studied in the absence and presence of the antibiotic fosfomycin by isothermal titration calorimetry. Fosfomycin binds covalently to MurA in the presence of UDPNAG and also in i ts absence as demonstrated by MALDI mass spectrometry. The covalent attachm ent of fosfomycin affects the thermodynamic parameters of UDPNAG binding si gnificantly: In the absence of fosfomycin the binding of UDPNAG is enthalpi cally driven (DeltaH = -35.5 kJ mol(-1) at 15 degreesC) and opposed by an u nfavorable entropy change (DeltaS = -25 J mol(-1) K-1). In the presence of covalently attached fosfomycin the binding of UDPNAG is entropically driven (DeltaS = 187 J mol(-1)K(-1) at 15 degreesC) and associated with unfavorab le changes in enthalpy (DeltaH = 28.8 kJ mol-1). Heat capacities for UDPNAG binding in the absence or presence of fosfomycin were -1.87 and -2.74 kJ m ol-1 K-1, respectively, indicating that most (approximate to 70%) of the co nformational changes take place upon formation of the UDPNAG-MurA binary co mplex. The major contribution to the heat capacity of ligand binding is tho ught to be due to changes in the solvent-accessible surface area. However, associated conformational changes, if any, also contribute to the experimen tally measured magnitude of the heat capacity. The changes in solvent-acces sible surface area were calculated from available 3D structures, yielding a DeltaC(p) of -1.3 kJ mol(-1) K-1; i.e., the experimentally determined heat capacity exceeds the calculated one. This implies that other thermodynamic factors exert a large influence on the heat capacity of protein-ligand int eractions.