Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity

To map the substrate specificity of cysteine proteases, two combinatorial p eptide libraries were synthesized and screened using the archetypal proteas e, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Re sults from the screening of library 2 indicated a preference for Pro or Val in the S-3 subsite and hydrophobic residues in S-2; the most prevalent res idue not being Phe but Val. The S-1 subsite exhibited a dual specificity fo r both small, nonpolar residues, Ala or Gly, as well as larger, Gin, and ch arged residues, Arg. Small residues predominated in the S-1'-S-4' subsites. Active peptides from the libraries and variations thereof were resynthesiz ed and their kinetics of hydrolysis by papain assessed in solution phase as says, Generally, there was a good correlation between the extent of substra te cleavage on solid phase and the k(cat)/K-M's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrat es being Y(NO2)PMPPLCTSMK(Abz) (k(cat)/K-M = 2109 (mM s)(-1)), Y(NO2)PYAVQS PQK(Abz) (k(cat)/K-M = 1524 (mM s)(-1)), and Y(NO2)PVLRQQRSK(Abz) (k(cat)/K -M = 1450 (mM s)(-1)). These results were interpreted in structural terms b y the use of molecular dynamics (MD). These MD calculations indicated two d ifferent modes for the binding of substrates in the narrow enzyme cleft.