Accelerated refolding of subtilisin BPN' by tertiary-structure-forming mutants of its propeptide.
The propeptide of subtilisin BPN', which functions as an intramolecular chaperone and a temporary inhibitor of subtilisin, is unique in that it acquires its three-dimensional structure by formation of a complex with the cognate protease. We previously showed that the successive amino acid replacements Ala47-->Phe, Gly13-->Ile, and Val65-->Ile in the propeptide to increase its hydrophobicity resulted in formation of a tertiary structure, accompanied by increased ability to bind to the protease and increased resistance to proteolysis. In this study, we examined the effects of these tertiary-structure-forming mutations on the intramolecular chaperone activity of the propeptide. The successive amino acid replacements mentioned above were introduced into pro-subtilisin*, possessing a Ser221-->Ala mutation in the catalytic residue. Refolding experiments were started by rapid dilution of the denatured pro-subtilisin*, and formation of tertiary structure in subtilisin was monitored kinetically by increase in tryptophan fluorescence. The wild-type pro-subtilisin* was found to refold with a rate constant of 4.8 x 10(-3) s(-1) in the equation describing an intramolecular process. The Ala47-->Phe replacement in the propeptide resulted in a 1.2-fold increase in the rate constant of subtilisin refolding. When the additional replacement Gly13-->Ile was introduced, refolding of subtilisin was substantially accelerated, and its kinetics could be fitted to a double exponential process composed of a fast phase with a rate constant of 2.1 x 10(-2) s(-1) and a slow phase with a rate constant of 4.5 x 10(-3) s(-1). The rate constant of the fast phase was increased slightly by a further replacement, Val65-->Ile. Since the slow phase is considered to correspond to proline isomerization, we concluded that tertiary-structure-forming mutations in the propeptide produce positive effects on its intramolecular chaperone activity through acceleration of the propeptide-induced formation of the tertiary structure of subtilisin BPN'.
Kojima
S
S
Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588, Japan. shuichi.kojima@gakushuin.ac.jp
Yanai
H
H
Miura
K
K
D019943:Amino Acid Substitution; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial; D014364:Tryptophan / Q000737:chemistry
D004792:Enzyme Precursors; D018832:Molecular Chaperones; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C057107:prosubtilisin; D014364:Tryptophan; D013381:Subtilisins