Search

Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP-conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30 100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP-conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.

Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.

Sequence conservation analysis relates the cholinesterases to a superfamily of polypeptides (Myers et al., 1988; Krejci et al., 1991), including enzymes such as microsomal carboxyesterase, cholesterol esterase, lysophospholipase, Geotrichum lipase and Drosophila esterase-6, as well as several noncatalytic polypeptides. AChE is the best characterized enzyme in this superfamily. Kinetic studies have indicated that the active site of AChE consists of two subsites: an anionic subsite to which the trimethylammonium group of acetylcholine binds and an esteratic subsite which interacts with the ester-bond region and mediates catalysis. Evidence also exists for an allosteric regulation of AChE activity by ligand binding to an anionic site(s) physically remote from the active site (Changeux, 1966).

An important step towards a comprehensive description of the structure-function properties of AChE has been made with the elucidation of the three dimensional structure of Torpedo californica AChE (TcAChE)1. Alignment of amino acid sequences of the cholinesterase family2 reveals over 56% sequence identity and 74% sequence homology1 between TcAChE and human AChE (HuAChE). The highest degree of conservation is in the active site “gorge” and in the residues which are probably involved in maintaining the overall structural integrity of the protein. These observations, together with the nearly identical activity profiles of the two enzymes led us to construct a HuAChE molecular model using the TcAChE structure as a template. Such models are useful for rationalizing the structural and functional effects of site directed mutagenesis3–6 as well as for probing the molecular basis for HuAChE reactivity. Here we provide another example for the use of this model for the analysis of the marked enantioselectivity exhibited by AChE towards organophophorous compounds7. In the case of isopropyl methylphosphonates (IMP) the ratio of bimolecular rate constants of inhibition (ki) for Sp-IMP vs Rp-IMP is in the range of 3 orders of magnitude’. This chiral selectivity is most likely a consequence of the steric requirements of the nucleophilic displacement reaction at the phosphorous atom in the enzyme environment. In order to test this assumption and also to test and refine our model, we have constructed the phosphonylation products of HuAChE, with Sp and Rp diastereoisomers of IMP.

A set of 41 overlapping peptides, representing the complete sequence of SFV-E2 protein were synthesized and analyzed in the ELISA test against murine anti-SFV sera. No single peptide was recognized by all antisera. Eight peptides were found to be highly reactive with hyperimmune anti-SFV sera. Six out of the eight peptide sequences coincide with the most hydrophilic regions of SFV-E2. Out of these, four peptides (amino acid positions 16–35, 61–80, 166–185, 286–305) that contain the least number of alphavirus conserved residues were selected. This panel constitutes the minimal number of peptides necessary and sufficient for specific recognition of hyperimmune mouse anti-SFV sera.

The generation and transport of the soluble glycoprotein (Gs) of wild-type vesicular stomatitis virus (VSV) were studied using cell fractionation and transport inhibitors. Gs was found in the rough endoplasmic reticulum (RER) and the Golgi-enriched membrane fractions of infected Chinese hamster ovary cells. The identity of intracellular Gs was confirmed by its precipitation with a monoclonal antibody to the ectodomain but not with a anti-peptide antibody directed against the first 15 amino acids at the carboxy terminus of the VSV transmembrane glycoprotein G. Their extracellular appearance was affected in a concentration-dependent manner by monensin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) and was completely inhibited by incubation at 20°. Inhibitors failed to dissociate the transport of Gs from G. These experiments indicate that in fibroblast cells Gs can be generated intracellularly, probably in the RER, and that Gs, like G, is transported from there to the Golgi complex and then presumably to the extracellular environment.