Aspartic proteinases — Fourier transform IR studies of the aspartic carboxylic groups in the active site of pepsin

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Abstract
Fourier transform (FTIR) difference spectra of pepsin minus diazoacetylnorleucine methyl ester (DAN) or minus diazoacetyl‐l‐phenylalanine methyl ester (DAP) modified pepsin, respectively, demonstrated that Asp‐215 is not deprotonated in pepsin. The FTIR difference spectrum of pepsin minus 1,2‐epoxyparanitrophenoxypropane (EPNP) modified pepsin demonstrates that Asp‐32 is present in pepsin as CO− 2 anion. The position of the v(C – O) vibration demonstrates that no (O … H … O)− hydrogen bond between Asp‐215 and Asp‐32 is formed. Furthermore, no H3O+ is present in the active center. Studies of the complex of pepsin with the inhibitor pepstatin prove that the inhibitor removes the water from the active site and Asp‐32 becomes protonated.