title: Engineering the oxyanion hole of trypsin for promoting the reverse of proteolysis.
abstract: Although proteases are capable of synthesizing peptide bonds via the reverse of proteolysis, they are not proficient at peptide fragment ligation. Further manipulations are needed to shift the native enzyme activity from the cleavage to the synthesis of peptides especially when longer peptides or even proteins are the target molecules of the reaction. This account reports on the synthetic potential of trypsin variants with engineered oxyanion holes mutated by proline mutations, which were designed to minimize proteolytic side reactions during peptide bond synthesis. From the six single and double proline-mutated trypsins, in particular, trypsinQ192P came out as the most promising biocatalyst enabling not only the ligation of cleavage-sensitive peptide fragments but also the selective N-terminal modification of a real protein substrate.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120, Halle, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Franke
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120, Halle, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Liebscher
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120, Halle, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Bordusa
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015202:Protein Engineering; D059748:Proteolysis; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics
chemical_list: D014357:Trypsin
keywords: biocatalysis; enzymatic peptide synthesis; enzyme engineering; protein modification; trypsin