Asp70 in the Peripheral Anionic Site of Human Butyrylcholinesterase

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Abstract
The goal of this work was to determine what amino acids at the mouth of the active‐site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady‐state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5–30‐fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though kcat, was the same for wild‐type and D70G mutant, being 24000 min−1 at low butyrylthiocholine concentrations (0.01–0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased threefold for wild type. The D70C mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active‐site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.