CATALYTIC MECHANISM OF THE ASPARTATE PROTEINASE PEPSIN-A - AN FTIR STUDY

The following FTIR difference spectra were studied: (pepsin) minus (As p 215 or Asp 32 modified pepsin), (pepsin + pepstatin) minus (the modi fied pepsin + pepstatin), (at 40 degrees C incubated pepsin + substrat e) minus (pepsin + substrate at 4 degrees C), and (at 40 degrees C inc ubated pepsin + substrate) minus (EPNP modified pepsin + substrate). F rom these spectra, it is concluded that in native pepsin Asp 215 is pr otonated and Asp 32 deprotonated. A water molecule is present between these Asp residues. When substrate is added, Asp 215 is deprotonated a nd Asp 32 becomes proton ated. This is performed by the hydrogen-bonde d system Asp 215-water-Asp 32. This system shows very large proton pol arizability due to collective proton motion. Asp 32 binds to the O ato m of the peptide group. The catalytic mechanism is a base catalysis pe rformed by the water molecule that is strongly polarized by the negati vely charged Asp 215 residue. With their lone pairs, the water molecul es attack the electrophilic carbon atom of the peptide group. (C) 1997 John Wiley & Sons, Inc.