Specificity of cathepsin B to fluorescent substrates containing benzyl side-chain-substituted amino acids at P1 subsite.

We have determined the kinetic parameters for the hydrolysis by cathepsin B of peptidyl-coumarin amide and intramolecularly quenched fluorogenic peptides with the general structures epsilonNH2-Cap-Leu-X-MCA and Abz-Lys-Leu-X-Phe-Ser-Lys-Gln-EDDnp, respectively. Abz (orthoaminobenzoic acid) and EDDnp (2,4-dinitrophenyl-ethylenediamine) are the fluorescent donor-acceptor pair, and X was Cys(SBzl), Ser(OBzl), and Thr(OBzl) containing benzyl group (Bzl) at the functional side chain of Cys, Ser, and Thr. The peptidyl-coumarin-containing Cys(SBz1), Ser(OBzl), and Thr(OBzl) have higher affinity cathepsin B, supporting the interpretation of the crystal structure of rat cathepsin B complexed with the inhibitor Z-Arg-Ser(OBzl)-CH2Cl that the benzyl group attached to Ser hydroxyl side chain occupies the enzyme S'(1) subsite [Jia et al. (1995), J. Biol. Chem. 270, 5527]. A similar effect of benzyl group was also detected in the internally quenched peptides. Finally, the benzyl group in substrates containing Cys(SBzl) amino acid at P1 seems to compensate the absence of adequate S2-P2 interaction in the hydrolysis of the peptides having Pro or Ala at P2 position.