Short, strong hydrogen bonds at the active site of human acetylcholinesterase: Proton NMR studies

Cholinesterases use a Glu-His-Ser catalytic triad to enhance the nucleophil icity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, s trong hydrogen bond (SSHB) between the Glu-His pair upon binding mechanism- based inhibitors, which form tetrahedral adducts, analogous to the tetrahed ral intermediates in catalysis [Viragh, C., et al. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14 .4 ppm assigned to an imidazole NH of the active-site histidine, but no des hielded proton resonances between 15 and 21 ppm. Addition of a 3-fold exces s of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)-trifluoroacet ophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the a ppearance of a broad, deshielded resonance of equal intensity with a chemic al shift delta of 17.8 ppm and a D/H fractionation factor phi of 0.76 +/- 0 .10, consistent with a SSHB between Glu and His of the catalytic triad. Fro m an empirical correlation of delta with hydrogen bond lengths in small cry stalline compounds, the length of this SSHB is 2.62 +/- 0.02 Angstrom, in a greement with the length of 2.63 +/- 0.03 Angstrom, independently obtained from phi. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, a nd subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 /- 0.02 and 2.65 -/+ 0.02 Angstrom respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to eith er Glu-327 of the catalytic triad or to Glu-199, also in the active site. C onformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonan ces of equal intensity at 16.5 and 15.8 ppm with phi values of 0.47 +/- 0.1 0 and 0.49 +/- 0.10 corresponding to average hydrogen bond lengths of 2.59 +/- 0.04 and 2.61 +/- 0.04 Angstrom, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pK(a) = 6.0 +/- 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 +/- 0.02 and 2.63 +/- 0.02 Angstrom, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 +/- 0.28 Angstrom) and sarin (2.53 +/- 0.26 Angstrom) and at low pH (2.52 +/- 0.25 Angstrom). Howe ver, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesteras e. and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part f rom the formation of a SSHB between Glu and His of the catalytic triad.