A Low-Barrier Hydrogen Bond in Subtilisin: 1H and 15N NMR Studies with Peptidyl Trifluoromethyl Ketones

Abstract

The Nδ1 proton of His 64 forms a hydrogen bond with Asp 32, as part of the catalytic triad in serine proteases of the subtilisin family. His 64 in subtilisin has been studied by ¹H and ¹⁵N NMR spectroscopy in the presence and absence of peptidyl trifluoromethyl ketones (TFMKs) that are transition state analog inhibitors. For subtilisin Carlsberg, the downfield resonance of the imidazolium Nδ1 proton is approximately 18.3 ppm and the D/H fractionation factor is 0.55 ± 0.04 at pH 5.5 (11 °C), and 0.63 ± 0.04 (5 °C) and 0.68 ± 0.04 at pH 6 (11 °C). In the complex between subtilisin Carlsberg and Z-l-leucyl-l-leucyl-l-phenylalanyltrifluoromethyl ketone (Z-LLF-CF₃) at pH values between 6.5 and 10.6, His 64 remains positively charged, and the D/H fractionation factor of its Nδ1 proton is 0.85 ± 0.05. In the complex between a subtilisin variant from Bacillus lentus and Z-LLF-CF₃, the proton resonance at 18.8 ppm is correlated with a ¹⁵N resonance at 197.6 ppm downfield from liquid NH₃ with a ¹J_NH of 81 Hz. The chemical shifts of subtilisin complexes with peptidyl TFMKs are among the most downfield shifts reported for any protein. At pH 9.5, His 64 is neutral and the D/H fractionation factor increases to 1.2 with a chemical shift of 15.0. His 64 is positively charged in the free enzyme at low pH, the inhibitor hemiketal complex at neutral pH, and the transition state for amide bond hydrolysis. These data thus provide indirect evidence for the presence of a low-barrier hydrogen bond in the catalytic mechanism of subtilisin proteases.