Insights into the Stereospecificity in Papain-Mediated Chemoenzymatic Polymerization from Quantum Mechanics/Molecular Mechanics Simulations

Chemoenzymatic peptide synthesis is an efficient and clean method to generate polypeptides for new applications in the fields of biomedical and functional materials. However, this enzyme-mediated synthesis is dependent on the reaction rate of the protease biocatalyst, which is essentially determined by the natural substrate specificity of the enzyme. Papain, one of the most studied cysteine proteases, is extensively used for the chemoenzymatic synthesis of new polypeptides. Similar to most proteases, papain displays high stereospecificity toward L-amino acids, with limited reactivity for the D-stereoisomer counterparts. However, the incorporation of D-amino acids into peptides is a promising approach to increase their biostability by conferring intrinsic resistance to proteolysis. Herein, we determined the stereospecific-limiting step of the papain-mediated polymerization reaction with the chiral substrates L/D-alanine ethyl ester (Ala-OEt). Afterward, we used Quantum Mechanics/Molecular Mechanics (QM/MM) simulations to study the catalytic mechanism at atomic level of detail and investigate the origin of its stereospecificity. The experimental and computational results show that papain is able to attack both L- and D-stereoisomers of Ala-OEt, forming an enzyme-substrate intermediate, and that the two reactions display a similar activation barrier. Moreover, we found that the reduced catalytic activity of papain in the polymerization of D-amino acids arises from the aminolysis step of the reaction, in which L-Ala-OEt displays a significantly lower free-energy barrier (12 kcal/mol) than D-Ala-OEt (30 kcal/mol). Further simulations suggest that the main factor affecting the polymerization of D-amino acids is the configuration of the D-acyl-intermediate enzyme, and in particular the orientation of its methyl group, which hinders the nucleophilic attack by other monomers and thus the formation of polypeptides.