title: Specificity of trypsin digestion and conformational flexibility at different sites of unfolded lysozyme.
abstract: Fourteen tryptic peptides and nine intermediates were identified as products of trypsin digestion of reduced and S-3-(trimethylated amino) propylated lysozyme. Kinetics of the appearance and disappearance of these products were observed by monitoring the peak areas on the chromatogram. In spite of the complicated reaction pathways, kinetics of the digestion of proteins and several intermediate products show simple decay curves with a single rate constant. In this paper, the trypsin susceptibility of the individual cleavage site is defined as a hydrolytic rate constant of the susceptible peptide bond in the presence of 10 nM trypsin. The cleavage sites of unfolded lysozyme are classified into two groups in terms of the trypsin susceptibility: one has a high susceptibility (10-20 h-1) and the other a low susceptibility (1.0-2.0 h-1). In the unfolded state of lysozyme, in conclusion, the region from residues 15 to 61 has a strong resistance to trypsin digestion; on the other hand, the C-terminal half of the polypeptide chain is flexible enough to fit into the active site of trypsin. In addition, six kinds of pentapeptides were synthesized as analogues of lysozyme fragments including Arg 14, Arg 21, Lys 33, Arg 45, Arg 61, and Arg 73. Kinetics of tryptic digestion of them were observed. Both kcat and KM were determined for these synthetic pentapeptides. The susceptibility of each cleavage site in pentapeptides is determined and compared with that corresponding in proteins. The susceptibility is usually higher when the susceptible peptide bond is included in proteins than in pentapeptides, so long as the conformation of peptide chain is flexible. However, susceptibilities of a few sites in proteins are lower than those in pentapeptides. This means that the peptide chains tend to fold locally to prevent trypsin from binding to the sites. It was found that the sites of Arg 21 and Arg 45 are indeed resistant to trypsin, but the site of Lys 33 is not so much, although the hydrolytic rate at Lys 33 itself is extremely slow.
authors.affiliation: Department of Physics, School of Science, Kwansei Gakuin University, Nishinomiya, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Noda
authors.forename: K
authors.initials: K
authors.lastname: Fujiwara
authors.forename: K
authors.initials: K
authors.lastname: Yamamoto
authors.forename: T
authors.initials: T
authors.lastname: Fukuno
authors.forename: S
authors.initials: S
authors.lastname: Segawa
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D009113:Muramidase; D014357:Trypsin