Papain-Catalyzed Peptide Bond Formation: Enzyme-Specific Activation with Guanidinophenyl Esters

• Published in: Chembiochem : a European journal of chemical biology 2011-09, Vol.12 (14), p.2201-2207
• Main Authors: de Beer, Roseri J. A. C., Zarzycka, Barbara, Amatdjais-Groenen, Helene I. V., Jans, Sander C. B., Nuijens, Timo, Quaedflieg, Peter J. L. M., van Delft, Floris L., Nabuurs, Sander B., Rutjes, Floris P. J. T.
• Format: Article
• Language: English
• Subjects: Biocatalysis; Biomimetic Materials - chemistry; Dipeptides - chemical synthesis; Dipeptides - chemistry; enzymatic peptide synthesis; Esters; Guanidine - chemistry; molecular dynamics; Molecular Dynamics Simulation; papain; Papain - metabolism; Peptides - chemical synthesis; Peptides - chemistry; Protein Conformation; Reproducibility of Results; substrate mimetics; Water - chemistry
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201100267

The substrate mimetics approach is a versatile method for small‐scale enzymatic peptide‐bond synthesis in aqueous systems. The protease‐recognized amino acid side chain is incorporated in an ester leaving group, the substrate mimetic. This shift of the specific moiety enables the acceptance of amino acids and peptide sequences that are normally not recognized by the enzyme. The guanidinophenyl group (OGp), a known substrate mimetic for the serine proteases trypsin and chymotrypsin, has now been applied for the first time in combination with papain, a cheap and commercially available cysteine protease. To provide insight in the binding mode of various Z‐XAA‐OGp esters, computational docking studies were performed. The results strongly point at enzyme‐specific activation of the OGp esters in papain through a novel mode of action, rather than their functioning as mimetics. Furthermore, the scope of a model dipeptide synthesis was investigated with respect to both the amino acid donor and the nucleophile. Molecular dynamics simulations were carried out to prioritize 22 natural and unnatural amino acid donors for synthesis. Experimental results correlate well with the predicted ranking and show that nearly all amino acids are accepted by papain. Fooling with substrate recognition: Guanidinophenyl (OGp) esters were utilized as potential substrate mimetics for papain‐induced dipeptide synthesis under aqueous conditions. Surprisingly, modeling studies instead revealed unprecedented enzyme‐specific activation, applicable to nearly all amino acids.