The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: Application of a combinatorial peptide library approach

The substrate specificity of CPB2.8 Delta CTE, a recombinant cysteine prote ase from Leishmania mexicana, was mapped by screening a fluorescence-quench ed combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S-3 subsite and for hydrophobic residues , both aliphatic and aromatic, in S-2. The S-1 subsite exhibited a specific ity for the basic residues Arg and Lys. Generally, the specificity of the p rimed subsites was less strict compared with the non-primed side which show ed preference for Arg, Lys and Ala in S-1, Arg, Pro and Gly in S-2 and Lys, Arg and Ser in S-4. By contrast, a strict preference for the basic residue s Arg and Lys was found for S-3. Overall, there was a trend for basic resid ues in alternating subsites and smaller residues in the primed sites compar ed with the non-primed sites. In addition, there were strict requirements f or the amino acids in subsites S-3-S-1. Fluorescence-quenched peptides from the library with the highest on-resin cleavage were resynthesised and the their kinetics of hydrolysis by CPB1.8 Delta CTE assessed in solution phase assays. Several good substances containing the quintessential dipeptide pa rticular to cathespin-L-like enzymes, -F-R/K-, in P-2 and P-1 were identifi ed (e.g. Y(NO2)-EKFR down arrow RGK-K(Abz)G, Abz = 2-aminobenzoyl; k(car)K( m) (1) = 4298 mM(-1) s(1)). However, novel substrates containing the dipept ide -L/I-Q- in P-2 and P-1 were also well hydrolysed (e.g. Y(NO2)-YLQ down arrow GIQK-K(Abz)G; k(cat)K(m)(-1) = 2583 mM(-1) s-(1)). The effect of util ising different fluorescent donor-quencher pairs on the value of k(cat)K(m) (-1) was examined. Generally, the use of the Abz/Q-EDDnp donor-quencher pai r (EDDnp = N-(2,4-dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO2) r esulted in higher k(cat)K(m)(-1) values for analogous substrates.