NMR Evidence for a Short, Strong Hydrogen Bond at the Active Site of a Cholinesterase

• Published in: Biochemistry (Easton) 2000-12, Vol.39 (51), p.16200-16205
• Main Authors: Viragh, Carol, Harris, Thomas K, Reddy, Putta Mallikarjuna, Massiah, Michael A, Mildvan, Albert S, Kovach, Ildiko M
• Format: Article
• Language: English
• Subjects: Acetophenones - metabolism; Acetophenones - pharmacology; Animals; Binding Sites - drug effects; Butyrylcholinesterase - chemistry; Butyrylcholinesterase - metabolism; Cholinesterase Inhibitors - metabolism; Cholinesterase Inhibitors - pharmacology; Enzyme Activation - drug effects; Horses; Hydrogen Bonding - drug effects; Kinetics; Nuclear Magnetic Resonance, Biomolecular - methods; Paraoxon - metabolism; Paraoxon - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0022644

Cholinesterases (ChE), use a Glu−His−Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp−His−Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576−4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by 1H NMR at 600 MHz and 25−37 °C. The downfield region of the 1H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, δ = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor φ = 0.72 ± 0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of δ with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64 ± 0.04 Å, in agreement with the length of 2.62 ± 0.02 Å independently obtained from φ. The addition of a 3-fold excess of m-(N,N,N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift δ = 18.1 ppm and a D/H fractionation factor φ = 0.63 ± 0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62 ± 0.04 Å from δ and 2.59 ± 0.03 Å from φ. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60 ± 0.22 and 2.66 ± 0.28 Å. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.