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.
Kobayashi
T
T
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Rutgers, Piscataway 08854.
Inouye
M
M
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