EXTREME STABILIZATION OF A THERMOLYSIN-LIKE PROTEASE BY AN ENGINEEREDDISULFIDE BOND

The thermal inactivation of broad specificity proteases such as thermo lysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have reve aled that a surface-located region in the N-terminal domain of the the rmolysin-like protease produced by Bacillus stearothermophilus is cruc ial for thermal stability. In this region a disulfide bridge between r esidues 8 and 60 was designed by molecular modelling, and the correspo nding single and double cysteine mutants were constructed. The disulfi de bridge was spontaneously formed in vivo and resulted in a drastic s tabilization of the enzyme. This stabilization presents one of the ver y few examples of successful stabilization of a broad specificity prot ease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and m utation of an area of the molecule involved in the partial unfolding p rocesses that determine thermal stability.