EVIDENCE FOR A LARGE DISPENSABLE SEGMENT IN THE SUBTILISIN-LIKE CATALYTIC DOMAIN OF THE LACTOCOCCUS-LACTIS CELL-ENVELOPE PROTEINASE

The Lactococcus lactis SK11 cell-envelope proteinase contains various inserts, located in external loops of the catalytic domain compared wi th related subtilisins. In this study, protein engineering was employe d to determine the function of the largest loop insertion (residues 23 8-388) relative to the subtilisin structure. By site-directed mutagene sis we have deleted the fragment of the proteinase gene encoding these 151 residues and analyzed the mutant Delta 238-388 proteinase for act ivity, (auto)processing and cleavage specificity. This extra segment i s found to be inessential for activity, and its removal does not inhib it folding as the mutant proteinase is still active. In addition, the N- and C-terminal autoprocessing of the Delta 238-388 proteinase appea rs to be unchanged. However, removal of residues 238-388 altered subst antially the caseinolytic specificity of the enzyme, indicating that t his extra segment influences substrate specificity. Residues 238-388 w ere shown to contain a specific epitope for a monoclonal antibody.