title: Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease.
abstract: The role of the C-terminal Leu300 in maintaining thermal stability of the neutral protease of Bacillus subtilis was investigated. From model building studies based on the three-dimensional structure of thermolysin, the neutral protease of B. thermoproteolyticus, it was concluded that this residue is located in a hydrophobic pocket composed of residues located in the C-terminal and the middle domain. To test the hypothesis that Leu300, by contributing to a stabilizing interaction between these domains, is important for enzyme stability, several neutral protease mutants were constructed and characterized. The thermostability of the enzyme was lowered by deleting Leu300 or by replacing this residue by a smaller (Ala), a polar (Asn) or a sterically unfavourable (Ile) amino acid. Thermostability was increased upon replacing Leu300 by Phe. These results are in agreement with model-building studies. The effects on thermostability observed after mutating the corresponding Val318 in the thermostable neutral protease of B.stearothermophilus were less pronounced.
authors: Department of Molecular Genetics, Centre of Biological Sciences, Haren, The Netherlands.
authors: V G
authors: VG
authors: Eijsink
authors: G
authors: G
authors: Vriend
authors: B
authors: B
authors: Van Den Burg
authors: G
authors: G
authors: Venema
authors: B K
authors: BK
authors: Stulp
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D015202:Protein Engineering; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D010450:Endopeptidases; D013820:Thermolysin