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Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

Abstract
A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.
AuthorsS Braxton, J A Wells
JournalBiochemistry (Biochemistry) Vol. 31 Issue 34 Pg. 7796-801 (Sep 01 1992) ISSN: 0006-2960 [Print] United States
PMID1510966 (Publication Type: Comparative Study, Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)
Chemical References
  • Peptide Fragments
  • Edetic Acid
  • Serine Endopeptidases
  • Subtilisins
  • thermitase
  • Calcium Chloride
  • Calcium
Topics
  • Amino Acid Sequence
  • Bacillus subtilis (genetics)
  • Base Sequence
  • Binding Sites
  • Calcium (metabolism)
  • Calcium Chloride (pharmacology)
  • Computer Simulation
  • Edetic Acid (pharmacology)
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Structure
  • Mutagenesis, Site-Directed
  • Peptide Fragments (chemistry, genetics, metabolism)
  • Serine Endopeptidases (chemistry)
  • Structure-Activity Relationship
  • Subtilisins (chemistry, genetics, metabolism)

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