Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule.
The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Rutgers, Piscataway 08854.
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D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D018833:Chaperonins; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011506:Proteins; C057107:prosubtilisin; D013381:Subtilisins; D018833:Chaperonins