A comparative study of the thermal inactivation of cytosolic and mitochondrial aspartate aminotransferases

Abstract

Rates of irreversible thermal inactivation of cytosolic and mitochondrial aspartate aminotransferases were measured over a large temperature range. Inactivation occurred by different kinetic pathways at high and low temperature with a transition point at about 60°C. This suggests that the isoenzymes exist in different conformations above and below that temperature. Discontinuities in plots of ln( V max) against 1/ T provided confirmatory evidence for this hypothesis. Activation parameters (Δ H ‡ and Δ S ‡) for the thermal inactivation process were calculated in the high and low temperature ranges. At high temperature the greater rate of inactivation of the mitochondrial isoenzyme is determined largely by a high value of Δ S ‡. This more than compensates for the fact that the Δ H ‡ is also greater for the mitochondrial isoenzyme indicative of greater intramolecular stabilising interactions compared with the cytosolic form. Thus the relative rates of inactivation are determined by the nature of the transition states rather than by intramolecular interactions in the folded proteins. At lower temperatures the kinetic stabilities of the isoenzymes reverse with the mitochondrial isoenzyme inactivating more slowly. This is largely because of a considerably smaller Δ S ‡ at low temperature which no longer compensates for the greater Δ H ‡ compared with the cytosolic isoenzyme.