A rational approach was taken to improve the stability of
subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early
autolysis products and amino-terminal sequence analysis. These studies showed that
subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164
peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced
autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of
subtilisin,
thermitase.
Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of
thermitase corresponding to residues 45-63 of
subtilisin BPN' was installed into
subtilisin BPN', the chimeric
protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This
enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in
EDTA. However, in 10 mM CaCl2, the mutant
subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type
subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from
thermitase were introduced near the second
autolysis loop in
subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type
subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of
autolysis can be stabilizing to a mesophilic
protease.