title: NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase.
abstract: 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 (1)H NMR at 600 MHz and 25-37 degrees C. The downfield region of the (1)H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, delta = 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 phi = 0.72+/-0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64+/-0.04 A, in agreement with the length of 2.62+/-0.02 A independently obtained from phi. 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 delta = 18.1 ppm and a D/H fractionation factor phi = 0.63+/-0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62+/-0.04 A from delta and 2.59+/-0.03 A from phi. 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 A. 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.
authors: Department of Chemistry, The Catholic University of America, Washington, DC 20064, USA.
authors: C
authors: C
authors: Viragh
authors: T K
authors: TK
authors: Harris
authors: P M
authors: PM
authors: Reddy
authors: M A
authors: MA
authors: Massiah
authors: A S
authors: AS
authors: Mildvan
authors: I M
authors: IM
authors: Kovach
mesh_terms: D000098:Acetophenones / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002091:Butyrylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006736:Horses; D006860:Hydrogen Bonding / Q000187:drug effects; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010261:Paraoxon / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000098:Acetophenones; D002800:Cholinesterase Inhibitors; C111602:m-(N,N,N-trimethylammonio)trifluoroacetophenone; D002091:Butyrylcholinesterase; D010261:Paraoxon