Non-productive binding of butyryl(thio)choline in the active site of vertebrate acetylcholinesterase.

The kinetic behavior of cholinesterases is unconventional. While their activities are higher than expected by classical Michaelis-Menten reaction mechanisms, at intermediate substrate concentrations they show strong inhibition by excess of substrate. To date, the main explanations used for all of their kinetic peculiarities include hindrance of product exit, entropically improved water orientation by a second substrate molecule, and complete blockade of the fully occupied active site. However, with the hydrolysis of butyryl(thio)choline by vertebrate acetylcholinesterase, there are time-dependent and substrate-concentration-dependent decreases in catalytic activity. As the substrate depletion results in the expected downwardly concave shape of the progress curves for product formation at low substrate concentrations, this cannot be the reason for the bending of the linear progress curves at higher substrate concentrations. A good theoretical and practical explanation was reached by including the time-dependent appearance of a non-productive enzyme-substrate complex in the reaction scheme. The slow establishment of this complex appears to be a rare occurrence of incorrect substrate orientation at the bottom of the active site, with this blocked by a second substrate molecule.

A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis.

A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63-64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species. Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M). All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs. Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans. Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine. A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo) limits the docking of butyrylcholine. This model is consistent with mutational analysis of the nematode enzymes. Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms. These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity.

DNA sequence of butyrylcholinesterase from the rat: expression of the protein and characterization of the properties of rat butyrylcholinesterase.

The rat is the model animal for toxicity studies. Butyrylcholinesterase (BChE), being sensitive to inhibition by some organophosphorus and carbamate pesticides, is a biomarker of toxic exposure. The goal of this work was to characterize the purified rat BChE enzyme. The cDNA sequence showed eight amino acid differences between the active site gorge of rat and human BChE, six clustered around the acyl binding pocket and two below the active site serine. A prominent difference in rat was the substitution of arginine for leucine at position 286 in the acyl pocket. Wild-type rat BChE, the mutant R286L, wild-type human BChE, and the mutant L286R were expressed in CHO cells and purified. Arg286 was found responsible for the resistance of rat BChE to inhibition by Triton X-100. Replacement of Arg286 with leucine caused the affinity for Triton X-100 to increase 20-fold, making it as sensitive as human BChE to inhibition by Triton X-100. Wild-type rat BChE had an 8- to 9-fold higher K(m) for the positively charged substrates butyrylthiocholine, acetylthiocholine, propionylthiocholine, benzoylcholine, and cocaine compared with wild-type human BChE. Wild-type rat BChE catalyzed turnover 2- to 7-fold more rapidly than human BChE, showing the highest turnover with propionylthiocholine (201,000 min(-1)). Human BChE does not reactivate spontaneously after inhibition by echothiophate, but rat BChE reactivates with a half-life of 4.3hr. Human serum contains 5mg/L of BChE and 0.01mg/L of AChE. Male rat serum contains 0.2mg/L of BChE and approximately 0.2mg/L of AChE.