Proton transfer processes in the hydrogen-bonded structure of the active centre of serine proteases — an FT—IR study

Abstract

FT—IR difference spectra of trypsin + inhibitor minus anhydrotrypsin + inhibitor complexes were used to study the proton transfer processes in the active centres of serine proteases. The modification of Ser 195 (chymotrypsinogen numbering) is clearly indicated by the difference spectra of trypsin minus anhydrotrypsin. This modification changes the environment of the Asp 102His 57 arrangement. At pH 3 the proton in the OH ⋯ N ⇌ O − ⋯ H +N hydrogen bond is shifted to Asp 102 with anhydrotrypsin whereas with trypsin a protonated His 57 residue is observed, indicating that in this case the equilibrium is completely shifted to the right hand side. Direct information on the catalytic mechanism is obtained by the bands of serine, histidine and aspartic acid in the difference spectra of trypsin + soybean trypsin inhibitor (SBTI) minus anhydrotrypsin + SBTI, as well as those of trypsin + bovine pancreatic trypsin inhibitor (BPTI) minus anhydrotrypsin + BPTI. In the case of the trypsin + SBTI complex a tetrahedral intermediate (TI)-like state is not formed at pH 3. It is, however, partially formed at pH 7. With the trypsin + BPTI complex the TI-like state is already partially formed at pH 3. At pH 7 the ES ⇌ TI equilibrium is shifted in favour of the right hand side. In both cases His 57 is protonated in this state. His 57 remains protonated in the TI due to the strong interaction of the Asp 102His 57 hydrogen bond with its environment since this hydrogen bond shows large proton polarizability. Thus, the serine −OH is not activated by a shift of the positive charge in a charge relay system. Instead an acid catalysis by the histidine residue is the true mechanism. It is discussed that the protonated histidine in the TI is favourable for the next step of the mechanism.