Resonance Raman and absorption spectroscopic characterization of the chemically engineered enzyme thiolsubtilisin; comparison with a natural thiolenzyme

The serine protease subtilisin Carlsberg has been converted to the cysteine protease thiolsubtilisin by chemically converting the active site Ser-221 to cysteine. The present experiments have been designed to explore the differences in the active site properties of thiolsubtilisin and papain - a natural cysteine protease. Resonance Raman and electronic absorption studies for the enzyme-substrate intermediates involving the acyl groups 5-methylthienylacryloyl and furylacryloyl have been used to probe the electrical effects of thiolsubtilisin's active site on the groups' delocalised π-electron systems. Unlike papain, thiolsubtilisin does not give rise to highly polarized π-electron systems in the bound substrates, characterized by low frequency ν_CC modes and red shifted λ_max's. However, for the 5-methylthienylacryloyl intermediate there is evidence for a minor photoinduced population which does have a polarized π-electron system. The results can be explained by differential binding of the substrate with respect to the α-helix dipole found in the active sites of thiolsubtilisin and papain.