INTRODUCTION OF DISULFIDE BONDS INTO BACILLUS-SUBTILIS NEUTRAL PROTEASE

The effects of engineered disulfide bonds on autodigestion and thermos tability of Bacillus subtilis neutral protease (NP-sub) were studied u sing site-directed mutagenesis. After modelling studies two locations that might be capable of forming disulfide bonds, both near previously determined autodigestion sites in NP-sub, were selected for the intro duction of cysteines. Analysis of mutant enzymes showed that disulfide bonds were indeed formed in vivo, and that the mutant enzymes were fu lly active. The introduced disulfides did not alter the autodigestion pattern of the NP-sub. All mutant NP-subs exhibited decreased thermost ability, which, by using reducing agents, was shown to be caused by th e introduction of the cysteines and not by the formation of the disulf ides. Mutants containing one cysteine exhibited intermolecular disulfi de formation at elevated temperatures, which, however, was shown not t o be the cause of the decreased thermostability. Combining the present data with literature data, it would seem that the introduction of dis ulfide bridges is unsuitable for the stabilization of proteases. Possi ble explanations for this phenomenon are discussed.