title: Unusual ligand binding at the active site domain of an engineered mutant of subtilisin BL.
abstract: As an attempt to recruit the third calcium binding site of thermitase into subtilisin BL, a Bacillus lentus alkaline protease (BLAP), the amino acid sequence from position 50 to 60 and position 92 was modified to the equivalent amino acids in thermitase. The resulting protein, designated BLAPm109, exhibited unusual biochemical features. Peptide mapping and gel electrophoresis revealed that two protein species co-purify in a ratio of about 1:1. Form 1 consisted of a single polypeptide of 269 amino acid residues. Form 2 was the same protein but with an internal peptide bond cleavage at the C-terminus of position 54. On electropherograms a dimer of Form 1 and Form 2 was also detectable. A zymogram showed that all three molecular species were catalytically active. From this protein mixture, crystals suitable for X-ray analysis were nevertheless obtained. SDS-PAGE of protein recovered from a crystal revealed that only Form 2 appears. in the crystal. The space group for this crystal was P21 with unit cell dimensions of a=42 angstroms, b=58 angstroms, c=47 angstroms and beta = 106.3 degrees. Examination of the preliminary electron density map revealed that the "thermitase loop" from 50 to 60 departs from the surface of the protein and winds through the active site of a symmetry-related copy of the asymmetric unit.
authors.affiliation: COGNIS, Inc., Santa Rosa, California 95407, USA.
authors.forename: C
authors.initials: C
authors.lastname: Paech
authors.forename: D W
authors.initials: DW
authors.lastname: Goddette
authors.forename: T
authors.initials: T
authors.lastname: Christianson
authors.forename: C R
authors.initials: CR
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D019281:Dimerization; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013381:Subtilisins / Q000235:genetics*
chemical_list: D013381:Subtilisins