title: Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis.
abstract: Using genetically engineered mutants of the neutral protease from Bacillus stearothermophilus (BsteNP), it had been shown that the surface-exposed structural motif constituted by Phe63 embedded in a four amino acid hydrophobic pocket is critical for the thermal stability of the thermophilic neutral proteases from Bacilli. To measure the stabilizing contribution of each hydrophobic interaction taking place between Phe63 and the hydrophobic pocket, we grafted this structural motif in the neutral protease from the mesophile Bacillus subtilis (BsubNP). This was accomplished by first creating the Thr63-->Phe mutant of BsubNP and then generating a series of mutants in which the four amino acids which in thermolysin surround Phe63 and form the hydrophobic pocket were added one after the other. By analysing the thermal stability of each mutant it was found that the 2 degrees C destabilizing effect of the Thr63-->Phe substitution was completely suppressed by the addition of the four amino acid hydrophobic pocket, each replacement providing a stabilizing contribution of approximately 0.8-1 degrees C. These results are discussed in the light of the peculiar mechanism of thermal inactivation of proteolytic enzymes.
authors.affiliation: Genetic Engineering and Microbiology Laboratories, ENIRICERCHE SpA, Milan, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Frigerio
authors.forename: I
authors.initials: I
authors.lastname: Margarit
authors.forename: R
authors.initials: R
authors.lastname: Nogarotto
authors.forename: V
authors.initials: V
authors.lastname: de Filippis
authors.forename: G
authors.initials: G
authors.lastname: Grandi
mesh_terms: D001412:Bacillus subtilis; D001426:Bacterial Proteins; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D008666:Metalloendopeptidases; C071298:bacillolysin