title: S' subsite mapping of serine proteases based on fluorescence resonance energy transfer.
abstract: A microassay based on fluorescence resonance energy transfer has been developed to determine the S' specificity of serine proteases. The protease-catalyzed acyl transfer from a fluorescing acyl donor ester to a P'1/P'2 variable hexapeptide library of nucleophiles labeled with a fluorescence quencher leads to an internally quenched peptide product and a fluorescent hydrolysis product. The amount of fluorescence quenching allows one to draw conclusions about the interaction of the nucleophile at the S' sites of the protease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The P'1/P'2 variable hexapeptide library with the general structure H-Xaa-Ala-Ala-Ala-Tyr(NO2)-Gly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gln and Glu, was prepared by solid-phase synthesis. Investigations of the S' specificity of trypsin, chymotrypsin and trypsin variants show that this assay is a fast and sensitive screening method for S' subsite mapping of serine proteases and is suitable for a high throughput screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis.
authors.affiliation: Faculty of Biosciences, Pharmacy and Psychology, Department of Biochemistry, University of Leipzig, Talstrasse 33, D-04103, Leipzig, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Grahn
authors.forename: T
authors.initials: T
authors.lastname: Kurth
authors.forename: D
authors.initials: D
authors.lastname: Ullmann
authors.forename: H D
authors.initials: HD
authors.lastname: Jakubke
mesh_terms: D001665:Binding Sites; D002918:Chymotrypsin / Q000737:chemistry; D004952:Esters / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods; D012697:Serine Endopeptidases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry
chemical_list: D004952:Esters; D019151:Peptide Library; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin