The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Site–directed mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core.