title: Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease.
abstract: On the basis of the homology with the Bacillus thermoproteolyticus zinc endopeptidase thermolysin, we hypothesized that Glu-143 and His-231 are the key residues for the catalytic activity of the Bacillus subtilis neutral protease. To test this possibility by site-directed mutagenesis, we substituted these two residues with Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively. All these substitutions dramatically affected the amount of secreted mutant proteins, as determined by immunological methods, and their catalytic activities. No appreciable secretion was observed with the three Glu mutants Trp, Ser and Arg, whereas the Glu----Ala mutant enzyme was secreted at a level of a few hundred micrograms per litre of culture. The His mutants were all secreted at higher levels (in the order of a few milligrams per litre) and their residual catalytic activity could be determined using Z-Ala-Leu-Ala as substrate. Our results confirm the key role played by Glu-143 and His-231 in catalysis and moreover suggest the existence of a relationship between the catalytic activity of the enzyme and the extent of its secretion. In this context, we present data suggesting an autoproteolytic mechanism of cleavage of the precursor form of the enzyme, analogous to the one previously reported for the B. subtilis subtilisin.
authors.affiliation: Molecular Biology, Laboratory, ENIRICERCHE S.p.A., Milan, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Toma
authors.forename: S
authors.initials: S
authors.lastname: Campagnoli
authors.forename: E
authors.initials: E
authors.lastname: De Gregoriis
authors.forename: R
authors.initials: R
authors.lastname: Gianna
authors.forename: I
authors.initials: I
authors.lastname: Margarit
authors.forename: M
authors.initials: M
authors.lastname: Zamai
authors.forename: G
authors.initials: G
authors.lastname: Grandi
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates; D006639:Histidine; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D015202:Protein Engineering
chemical_list: D005971:Glutamates; D006639:Histidine; D010447:Peptide Hydrolases