Probing the specificity of cysteine proteinases at subsites remote from the active site:: analysis of P4, P3, P2′ and P3′ variations in extended substrates

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S-4 and S-3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S-2' and S-3'. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P-4, P-3, P-2' and P-3' were made. The S-4 to S-2' subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S-3', indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S-4, S-3, S-3' and S-3' of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P-2' respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P-3'. Basic residues at P-3 and p(4) were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P-2' (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His(111) (H111A) and His(110) (H110A) of cathepsin B led to an increase in k(cat) values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.