The role of Tyr71 in Streptomyces trypsin on the recognition mechanism of structural protein substrates.
Studies of substrate recognition by serine proteases have focused on specificities at the primary S1-Sn sites, but topological specificities (i.e. recognition at distinct three-dimensional structural motifs) have not been established. This is the first report to identify the key amino acid residue conferring topological specificity. A serine protease from Streptomyces omiyaensis (SOT), which is a trypsin-like enzyme, was chosen as a model enzyme to clarify the recognition mechanism of structural protein substrates in serine proteases. We have found previously that the topological specificities of SOT and S. griseus trypsin (SGT) for high molecular mass substrates differ greatly, even though the enzymes have similar primary structures. In this study, we constructed chimeras between SOT and SGT using an in vivo DNA shuffling system and several mutants to identify the key residues involved in topological specificities. By comparing the substrate specificities of chimeras and mutants, we found that residue 71 of SOT, which is separate from the catalytic triad, contributes to the topological specificity. Using site-directed mutagenesis, residue 71 of SOT was also found to be crucial for catalytic efficiency and enzyme conformation.
Uesugi
Yoshiko
Y
Research Institute for Biological Sciences, Okayama, Japan.
Usuki
Hirokazu
H
Iwabuchi
Masaki
M
Hatanaka
Tadashi
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002417:Cattle; D024042:Collagen Type I / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013305:Streptomyces griseus / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D024042:Collagen Type I; D024141:Collagen Type IV; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D014357:Trypsin