Rendering One Autolysis Site in Bacillus Subtilis Neutral Protease Resistant to Cleavage Reveals a New Fission

• Published in: Biotechnology and applied biochemistry 1998-04, Vol.27 (2), p.125-132
• Main Authors: Burg, Bertus, Eijsink, Vincent G. H., Vriend, Gert, Veltman, Oene R., Venema, Gerard
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacillus subtilis - enzymology; Binding Sites; Biological and medical sciences; Biotechnology; Enzyme Stability; Fundamental and applied biological sciences. Psychology; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metalloendopeptidases - metabolism; Methods. Procedures. Technologies; Models, Molecular; Mutation; Protein Conformation; Protein engineering; Sequence Analysis; Substrate Specificity
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1111/j.1470-8744.1998.tb01384.x

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