Kinetic study on the irreversible thermal denaturation of yeast phosphoglycerate kinase.
Differential scanning calorimetry transitions for the irreversible thermal denaturation of yeast phosphoglycerate kinase at pH 7.0 are strongly scanning-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. To test this possibility, we have carried out a kinetic study on the thermal inactivation of the enzyme. The inactivation kinetics are comparatively fast within the temperature range of the calorimetric transitions and can be described phenomenologically by the equation dC/dt = -alpha C2/(beta + C), where C is the concentration of active enzyme at a given time, t, and alpha and beta are rate coefficients that depend on temperature. This equation, together with the values of alpha and beta (within the temperature range 50-59 degrees C) have allowed us to calculate the fraction of irreversibly denatured protein versus temperature profiles corresponding to the calorimetric experiments. We have found that (a) irreversible denaturation takes place during the time the protein spends in the transition region and (b) there is an excellent correlation between the temperatures of the maximum of the calorimetric transitions (Tm) and the temperatures (Th) at which half of the protein is irreversibly denatured. These results show that the differential scanning calorimetry transitions for the denaturation of phosphoglycerate kinase are highly distorted by the rate-limited irreversible process. Finally, some comments are made as to the use of equilibrium thermodynamics in the analysis of irreversible protein denaturation.
Galisteo
M L
ML
Departamento de Quimica Fisica (Facultad de Ciencias) e Instituto de Biotecnologia, Universidad de Granada, Spain.
Mateo
P L
PL
Sanchez-Ruiz
J M
JM
D002152:Calorimetry, Differential Scanning; D006358:Hot Temperature; D007700:Kinetics; D008433:Mathematics; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013816:Thermodynamics
D010735:Phosphoglycerate Kinase