Manipulating the autolytic pathway of a Bacillus protease

Autolytic degradation of Bacillus subtilis thermolysin-like proteinase (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously, we reported five autolysis sites in B. subtilis neutral protease (Van den Burg et al., 1990, Biochem. J. 272:93-97). In an attemp to render the enzyme less susceptible to autolytic breakdown, one of the sites, located between Leu156 and Ile157, was modified by substituting the residue C-terminal of the fission (P-1' residue), Ile157, by Asp. The introduction of the Asp, which is unfavoured at the P-1' position in substrates for TLPs, resulted in an altered autolysis pattern. The peptides formed in wild-type TLP-sub by chain cleavage N-terminal of Ile157, were absent in the constructed mutant (I157D). However, an autolysis product was detected with I157D that was not seen in case of the wild-type. The N-terminal sequence of this peptide was determined and it was concluded that in the mutant a peptide accumulated as the result of chain cleavage of the Gly148-Val149 bond. Structural aspects and effects of the mutation on the activity have been analyzed.