RENDERING ONE AUTOLYSIS SITE IN BACILLUS-SUBTILIS NEUTRAL PROTEASE RESISTANT TO CLEAVAGE REVEALS A NEW FISSION

Autolytic degradation of the thermolysin-like proteinase of Bacillus s ubtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in Tip-sub [Van den Burg et al, (1990) Biochem. J. 272, 93-97]. In an attempt to render the enzyme less susceptible to autoly tic breakdown, one of the fission sites, located between Leu-156 and I le-157, was modified by replacing Ile-157, C-terminally located with r espect to the fission site, by an Asp residue. Aspartic acid is less p referred at this position with respect to the substrate preference of TLP-sub, Modelling studies indicated that this mutation was unlikely t o cause conformational changes in the enzyme. Although the 156-157 fis sion was not observed in the mutant enzyme, a new fission site, betwee n Gly-148 and Val-149, was now observed.