Identification of oxygen nucleophiles in tetrahedral intermediates: 2H and 18O induced isotope shifts in 13C NMR spectra of pepsin-bound peptide ketone pseudosubstrates

Abstract

The synthetic ketone peptide analogue of pepstatin, isovaleryl-L-valyl-[3- 13C]-(3-oxo-4S)-amino-6-methylheptanoyl-L-alanyl-isoamylamide is a strong inhibitor of aspartyl proteases. When the peptide is added to porcine pepsin in H 2O at pH 5.1, the 13C NMR chemical shift of the ketone carbon moves from 208 ppm for the inhibitor in solution to 99.07 ppm when bound to the enzyme active site. In 2H 2O the bound shift is 98.71 ppm, 0.36 ppm upfield. For the analogous experiment contrasting H 2 16O and H 2 18O, the 13C chemical shift was 0.05 ppm to higher field for the heavier isotope. These data show that water, and not an enzyme nucleophile, adds to the peptide carbonyl to yield a tetrahedral diol adduct in the enzyme-catalyzed reaction, and provide a method for differentiating between covalent and non-covalent mechanisms.