The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu¹³-Ala¹⁴, Leu¹⁷-Val¹⁸ and Phe²⁵-Tyr²⁶; minor cleavage sites are the bonds Asn³-Gln⁴, Ser⁹-His¹⁰ and Leu¹⁵-Tyr¹⁶. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers small neutral amino acid residues in the subsites S₂ and S₃, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P₂ and P₃ positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH₂F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Val-Phe-CH₃ and Z-Phe-Lys(Z)-CH₃ inhibit competitively both cathepsin S and cathepsin L in the micromolar range.