Thermal responses of mutant enzymes and temperature limits to growth

Summary

The half-lives of fifty-two mutant forms of β-galactosidase, in which serine had been substituted for the original amino-acid, were measured at 57°C. Sixty percent of the mutant enzymes had less than half the stability of the normal enzyme at high temperature but none had greater sensitivity to low temperature. A sample of these thermolabile enzymes showed unaltered activation energy. Enzymes with altered substrate affinity generally had reduced apparent heats of activation, decreased heats and entropies of association, and were thus relatively less sensitive to temperature change.

The intracellular behaviour of thermolabile enzymes was examined by showing that substrate hydrolysis is the limiting step in lactose utilization, that the enzyme-substrate complex is more stable than the free enzyme and that binding of the enzyme to structures within the cell increases stability and lowers affinity. Amongst enzymes having half-lives less than about seven minutes at 47.75°C, there is a close relationship between enzyme lability and diminished growth rate on lactose at 44°C. The effect of mutation on the thermal behaviour of multi-enzyme systems and the relation of enzyme thermolability to temperature-induced growth deficiencies and temperature-dependent heterosis are discussed.