Thermodynamic and kinetic stability of penicillin acylase from Escherichia coli

Thermal denaturation of penicillin acylase (PA) from Escherichia coli has been studied by high-sensitivity differential scanning calorimetry as a function of heating rate, pH and urea concentration. It is shown to be irreversible and kinetically controlled. Upon decrease in the heating rate from 2 to 0.1 K min− 1 the denaturation temperature of PA at pH 6.0 decreases by about 6 °C, while the denaturation enthalpy does not change notably giving an average value of 31.6 ± 2.1 J g− 1. The denaturation temperature of PA reaches a maximum value of 64.5 °C at pH 6.0 and decreases by about of 15 °C at pH 3.0 and 9.5. The pH induced changes in the denaturation enthalpy follow changes in the denaturation temperature. Increasing the urea concentration causes a decrease in both denaturation temperature and enthalpy of PA, where denaturation temperature obeys a linear relation. The heat capacity increment of PA is not sensitive to the heating rate, nor to pH, and neither to urea. Its average value is of 0.58 ± 0.02 J g− 1 K− 1. The denaturation transition of PA is approximated by the Lumry-Eyring model. The first stage of the process is assumed to be a reversible unfolding of the α-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the β-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the α-subunit, and a rate constant of the irreversible stage are determined as a function of pH and urea concentration. Structural features of the folded and unfolded conformation of the α-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.