ANALYSIS OF STRUCTURAL DETERMINANTS OF THE STABILITY OF THERMOLYSIN-LIKE PROTEASES BY MOLECULAR MODELING AND SITE-DIRECTED MUTAGENESIS

The thermolysin-like protease (TLP) produced by Bacillus stearothermop hilus CU21 (TLP-ste) differs at 43 positions from the more thermally s table thermolysin (containing 316 residues in total), Of these differe nces, 26 were analysed by studying the effect of replacing residues in TLP-ste by the corresponding residues in thermolysin, Several stabili zing mutations were identified but, remarkably, considerable destabili zing mutational effects were also found, A Tyr-rich three residue inse rtion in TLP-ste (the only deletional/insertional difference between t he two enzymes) appeared to make an important contribution to the stab ility of the enzyme. Mutations with large effects on stability were al l localized in the beta-pleated N-terminal domain of TLP-ste, confirmi ng;observations that this domain has a lower intrinsic stability than the largely alpha-helical C-terminal domain, Rigidifying mutations suc h as Gly58 --> Ala and Ala69 --> Pro were among the most stabilizing o nes, Apart from this observation, the analyses did not reveal general rules for stabilizing proteins. Instead, the results highlight the imp ortance of context in evaluating the stability effects of mutations.