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 degrees 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 degrees C at pH 6.0 and decreases by about of 15 degrees 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 alpha-subunit. It activates the second stage involving dissociation of two subunits and subsequent denaturation of the beta-subunit. This stage is irreversible and kinetically controlled. Using this model the temperature, enthalpy and free energy of unfolding of the alpha-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 alpha-subunit as well as of the transition state of the PA denaturation in aqueous and urea solutions are discussed.
Grinberg
Valerij Ya
VY
N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Vavilov Street 28, 119991, Moscow, Russia.
Burova
Tatiana V
TV
Grinberg
Natalia V
NV
Shcherbakova
Tatiana A
TA
Guranda
Dorel T
DT
Chilov
Ghermes G
GG
Svedas
Vytas K
VK
D002152:Calorimetry, Differential Scanning; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010399:Penicillin Amidase / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D021122:Protein Subunits / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D021122:Protein Subunits; D014508:Urea; D010399:Penicillin Amidase