Revisiting the S2 specificity of papain by structural analogs of Phe.
Papain characteristically has a strong preference for encoded L-aromatic amino acids (Phe > Tyr) at P2 position. We re-examined papain S2 specificity using structural analogs of Phe, in fluorogenic substrates of the series: dansyl-Xaa-Arg-Ala-Pro-Trp (Xaa = P2 residue). Kinetic analyses showed that the S2 pocket accommodates a broad spectrum of Phe derivatives. Papain is poorly stereoselective towards Dns-(D/L)-Phe-Arg-Ala-Pro-Trp and binding is not critically affected by replacement of the benzyl ring by the non-aromatic lateral chain of cyclohexylalanine. The Km was significantly improved by mono- and di-chlorination of Phe, or by its substitution by an electronegative group-like NO2, but the specificity constant was unchanged. Shortening or lengthening the side chain by adding or removing a methylene group impairs the P2/S2 interactions significantly, as do constrained structural analogs of Phe. Incorporation of benzyl-substituted phenylalanyl amino acid could help to design peptide-derived inhibitors with greater affinity and bioavailability.
Lecaille
F
F
Laboratory of Enzymology and Protein Chemistry, Faculty of Medicine, University François Rabelais, Tours, France.
Serveau
C
C
Gauthier
F
F
Lalmanach
G
G
D001665:Binding Sites; D010206:Papain / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives*; D013379:Substrate Specificity
D010649:Phenylalanine; D010206:Papain