Conjugating Catalytic Polyproline Fragments with a Self-Assembling Peptide Produces Efficient Artificial Hydrolases

A polyproline fragment containing a catalytic dyad of His-His or Ser-His was coupled with a self-assembling peptide MAX1 to design new hydrolases (H2H5 and H2S5) for catalyzing ester hydrolysis. Circular dichroism measurements indicated that the peptides change their conformation from random coils to β-sheets when pH increases from 5 to 10. IR spectra also displayed the vibration modes corresponding to their β-structures at pH 9.0. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) measurements showed that in solution, the designed peptides self-assemble into network fibrils having a significantly increased catalytic efficiency on ester hydrolysis. On p-nitrophenyl acetate (p-NPA) substrate, the designed peptides exhibit high catalytic efficiency at pH 9.0 (k_cat/K_M = 12.1 M^-1 s^-1 for H2H5, 13.3 M^-1 s^-1 for H2S5), and their efficiency is even better at pH 10.0 (k_cat/K_M = 24.3 M^-1 s^-1 for H2H5, 99.4 M^-1 s^-1 for H2S5). Additionally, H2H5 and H2S5 also display good activity on catalyzing the hydrolysis of p-nitrophenyl-(2-phenyl)-propanoate (p-NPP) and p-nitrophenyl methoxyacetate (p-NPMA). Combining the polyproline-based catalytic scaffold with a self-assembling peptide generates an efficient hydrolase, providing a new design for effective artificial enzymes.