S ' subsite mapping of serine proteases based on fluorescence resonance energy transfer

A microassay based on fluorescence resonance energy transfer has been devel oped to determine the S' specificity of serine proteases. The protease-cata lyzed acyl transfer from a fluorescing acyl donor ester to a P-1'/P-2' vari able hexapeptide library of nucleophiles labeled with a fluorescence quench er leads to an internally quenched peptide product and a fluorescent hydrol ysis product. The amount of fluorescence quenching allows one to draw concl usions about the interaction of the nucleophile at the S' sites of the prot ease. o-Aminobenzoic acid and 3-nitrotyrosine were used as an efficient don or-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)-G ly-OH and H-Ala-Xaa-Ala-Ala-Tyr(NO2)-Gly-OH, where Xaa represents Arg, Lys, Met, Phe, Ala, Gly, Ser, Gin and Glu, was prepared by solid-phase synthesi s. Investigations of the S' specificity of trypsin, chymotrypsin and trypsi n variants show that this assay is a fast and sensitive screening method fo r S' subsite mapping of serine proteases and is suitable for a high through put screening. The assay might be useful for the development of restriction proteases and the estimation of yields in enzymatic peptide synthesis. (C) 1999 Elsevier Science B.V. All rights reserved.