Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides Available to Purchase

We have examined in detail the specificity of the subsites S₁, S₂, S₁′ and S₂′ for the carboxydipeptidase activity of cathepsin B by synthesizing and assaying four series of internally quenched fluorescent peptides based on the sequence Dnp-GFRFW-OH, where Dnp (2,4-dinitrophenyl) is the quenching group of the fluorescence of the tryptophan residue. Each position, except the glycine, was substituted with 15 different naturally occurring amino acids. Based on the results we obtained, we also synthesized efficient and sensitive substrates that contained o-aminobenzoic acid and 3-Dnp-(2,3-diaminopropionic acid), or ∊-amino-Dnp-Lys, as the fluorescence donor—receptor pair. The higher kinetic parameter values for the carboxydipeptidase compared with the endopeptidase activity of cathepsin B allowed an accurate analysis of its specificity. The subsite S₁ accepted preferentially basic amino acids for hydrolysis; however, substrates with phenylalanine and aliphatic side-chain-containing amino acids at P₁ had lower K_m values. Despite the presence of Glu²⁴⁵ at S₂, this subsite presented clear preference for aromatic amino acid residues, and the substrate with a lysine residue at P₂ was hydrolysed better than that containing an arginine residue. S₁′ is essentially a hydrophobic subsite, and S₂′ has particular preference for phenylalanine or tryptophan residues.