Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission.
Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in TLP-sub [Van den Burg et al. (1990) Biochem. J. 272, 93-97]. In an attempt to render the enzyme less susceptible to autolytic breakdown, one of the fission sites, located between Leu-156 and Ile-157, was modified by replacing Ile-157, C-terminally located with respect to the fission site, by an Asp residue. Aspartic acid is less preferred at this position with respect to the substrate preference of TLP-sub. Modelling studies indicated that this mutation was unlikely to cause conformational changes in the enzyme. Although the 156-157 fission was not observed in the mutant enzyme, a new fission site, between Gly-148 and Val-149, was now observed.
Van den Burg
B
B
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Eijsink
V G
VG
Vriend
G
G
Veltman
O R
OR
Venema
G
G
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D004795:Enzyme Stability; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017421:Sequence Analysis; D013379:Substrate Specificity
D008666:Metalloendopeptidases; C044194:microbial metalloproteinases