8.2.2.1 Physicochemical Properties of Acetylcholinesterase
The purification of AChE and the elucidation of its physicochemical properties were
achieved by using the enzyme isolated from the electric eel (Electrophorus electroplax)
the richest source of AChE, as well as from brain and erythrocytes. With high ionic
strength solutions (1 M NaCl or 2 M MgCl 2 ), the extraction is selective and can be facil-
itated by treatment of the electroplax with collagenase. The basic unit of the enzyme is
a tetramer with a molecular weight of 320,000; each of the protomers contains an active
site. Normally, three such tetrameric units are linked through disulfide bonds to a
50 ×2 nm stem. This stem is a collagen triple helix that seems to have a structural role
only. In neural and electroplax tissue, the enzyme “trees” (stems with tetramers) are
anchored in the basement membrane or neurolemma—a porous, collagen-rich struc-
ture. However, because many tissues (erythrocytes, peripheral ganglia) do not have a
basement membrane, attachment of the AChE must occur in another, unknown fashion.
Analysis of the amino acid composition of the enzyme shows that it bears a close
similarity to the AChR in its high proportion of acidic amino acids.
In contrast to the AChR, AChE does not bind bungarotoxin or sulfhydryl reagents. It
is inhibited by excess substrate (3 × 10 −^6 M), and the KDof electric eel AChE is about
10 −^4 M. The specific activity of the enzyme is one of the highest known: 750 nmol/mg-hr,
with a turnover time of 30–60 msec and a turnover number of 2–3 ×106. It is therefore
one of the most efficient and fastest enzymes known.
The active site of AChE has the composition of most serine esterases. It includes a
charge-relay system of histidine and serine, and an acidic center, probably glutamate,
which binds the choline cation. The serine hydroxyl is rendered more nucleophilic
through the proton-acceptor role of histidine and is capable of executing a nucleophilic
attack on the carbonyl carbon of ACh. A tetrahedral transition state is reached, result-
ing in serine acetylation and the desorption of free choline. The acetyl group is taken
over by histidine as an N-acetate, which is then easily hydrolyzed, regenerating the
enzyme active site. The choline is taken up into the nerve ending by an active transport
system and reused for ACh synthesis. Finally, the acetate goes into the ubiquitous
acetate pool of intermediary metabolism.
On the basis of studies of the hydrolysis rate of succinyl-methylcholine isomers,
Stenlake proposed the hypothesis that the AChR and AChE bind two different faces of
ACh, which implies that they have opposite sterochemistries at their respective anionic
subsites. Simultaneous attachment to the receptor and the enzyme is therefore impossi-
ble, although a fast sequential attack of the enzyme on the receptor-bound neurotrans-
mitter is still feasible since no ACh reorientation is necessary for this. However, there
is no evidence that such a sequence of events occurs in vivo.
Another cholinesterase, called serum cholinesterase or butyrylcholinesterase, is
found in serum and the liver. It plays an important role in drug metabolism.
8.2.2.2 Anticholinesterase Inhibitors—General Properties
Anticholinesterase drugs are compounds that block AChE and inhibit the destruction of
released ACh. The resultant higher neurotransmitter levels then increase the biological
response. Anticholinesterases can therefore be considered as indirect cholinergic
agents. Acetycholinesterase inhibitors can act by either of two mechanisms:
ENDOGENOUS MACROMOLECULES 487