Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.
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.
Braxton
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S
Protein Engineering Department, Genetech, Inc., South San Francisco, California 94080.
Wells
J A
JA
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D003198:Computer Simulation; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D010446:Peptide Fragments; D004492:Edetic Acid; D012697:Serine Endopeptidases; D013381:Subtilisins; C022728:thermitase; D002122:Calcium Chloride; D002118:Calcium