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.

Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad

“…As shown in Fig. 5, there is no signal in the 13-22-ppm range in the absence of a (45)(46)(47)50). Besides the two strong hydrogen bonds of the triad, there are two additional short hydrogen bonds between the SHCHC ligand and the side chains of Arg 90 and Ser 86 in the MenH-SHCHC structure that are 2.66 and 2.56 Å, respectively.…”

“…When these enzymes interact with their mechanism-based inhibitors, the imidazole ring in the side chain of the central histidine is protonated, and its hydrogen bond with the acidic residue is greatly strengthened, leading to a significant downfield shift of the corresponding proton signal into the 16 -19.5-ppm range (45)(46)(47). Based on these facts, we used NMR spectroscopy to detect the proton in the strong hydrogen bond between the side chains of Asp 201 and His 232 in the catalytic triad of 15 N-labeled MenH.…”

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

“…23 The results of the present study raise concern about assignment of downfield NMR peaks solely based on sitedirected mutagenesis experiments or by simple comparison with serine proteases, which is commonly adopted by others. 24,25,32,33 Even among serine proteases, it has been reported that the prolyl oligopeptidase family exhibits important dissimilarities in the active site compared to the pancreatic and subtilisin classes of serine proteases. 34 In the case of the prolyl oligopeptidase, non-catalytic histidine imidazole ring-attached protons are engaged in relatively strong hydrogen bonds based on the observation that their downfield chemical shifts are virtually independent of pH up to pH 9.5.…”

Observation of a Short, Strong Hydrogen Bond in the Active Site of Hydroxynitrile Lyase from Hevea brasiliensis Explains a Large pK Shift of the Catalytic Base Induced by the Reaction Intermediate

“…The occurrence of SSHBs has so far been reported for a considerable number of enzymes, including ketosteroid isomerase (15)(16)(17), triosephosphate isomerase (18), serine proteases (3,6,19), tryptophan synthase (20), 2-amino-3-ketobutyrateCoA ligase (21), and cholinesterases (22,23). Their occurrence has typically been suggested by NMR spectroscopy (24,25).…”