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

Abstract

The N delta 1 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 N delta 1 proton is approximately 18.3 ppm and the D/H fractionation factor is 0.55 +/- 0.04 at pH 5.5 (11 degrees C), and 0.63 +/- 0.04 (5 degrees C) and 0.68 +/- 0.04 at pH 6 (11 degrees 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 N delta 1 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 1JNH 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.

The Catalytic Role of Aspartate in a Short Strong Hydrogen Bond of the Asp274–His32 Catalytic Dyad in Phosphatidylinositol-specific Phospholipase C Can Be Substituted by a Chloride Ion

“…The 15 N chemical shift was determined to be 180 Ϯ 10 ppm by a series of continuous wave decoupling experiments (spectra not shown). This 15 N chemical shift value is close to the typical value for a ϭ NH ϩ in model compounds (ϳ175 ppm) (10,11), suggesting that His 32 is largely positively charged. The protonation state of His 32 at pH 5.0 is consistent with its pK a value described in the next section.…”

Oxyanion and tetrahedral intermediate stabilisation by subtilisin: Detection of a new tetrahedral adduct

“…The absence of a signal at ~ 15 p.p.m. due to the N δ 1 proton of imidazole [30] and the presence of the signals at ~13 and ~19 p.p.m. due to the N δ 1 and N ε 2 protons of the imidazolium ion of histidine-64 shows that when the glyoxal inhibitor is bound to subtilisin the active site histidine residue (Histidine-64) is fully protonated and its pK a is greater than 10.5 (Fig.…”

A Low-barrier Hydrogen Bond Between Histidine of Secreted Phospholipase A2 and a Transition State Analog Inhibitor

“…It has been suggested that in chymotrypsin and subtilisin complexes with inhibitors the active site histidine is only partially charged (between 0.5 and 1.0) due to the depolarization of LBHB, 2,3,25 as if the bridging proton is partially (about 20 -30%) transferred from the donor (active site His) to the acceptor (active site Asp). However, the only experimental evidence presented is the Here we present a more comprehensive analysis of both imidazole NH chemical shifts in comparisons with those in free enzyme as well as in model compounds, which strongly supports a partially positively charged H48 in the sPLA 2 -HK32 complex.…”