The neuromuscular junction, (a) Connection between a nerve and a muscle
fiber, (b) Invagination of a nerve terminal into the muscle fiber. The
acetylcholine receptor-rich domains are located on the crest of the fold.
an esteratic site and a site consisting of a negative charge
(Figure 6-9).
The esteratic site contains a seryl hydroxyl group whose
nucleophilicity toward the carbonyl carbon of the sub-
strate is enhanced by an appropriately located imidazole
group of histidine that functions as a general base cat-
alyst. A side-chain carboxyl group that is suitably lo-
cated apparently functions to hold the imidazole and
imidazolium ion in place. The substrate is positioned on
the enzyme so that its positively charged nitrogen atom
is attracted to the negatively charged active site of the
enzyme by both coulombic and hydrophobic forces. The
acyl carbon of the substrate is subjected to nucleophilic at-
tack by the oxygen atom of the serine hydroxyl group. This
catalytic mechanism is similar to that of other serine hy-
drolases (esterases and the proteinases, i.e., chymotrypsin,
trypsin, elastase, and thrombin). Although they have dif-
ferent functions, these enzymes have a common catalytic
mechanism, which supports the view that they evolved
from a common ancestor.
The inactive phosphorylated acetylcholinesterase un-
dergoes hydrolysis to yield free enzyme, but the reac-
tion is extremely slow. However, a nucleophilic reagent
(e.g., hydroxylamine, hydroxamic acids, and oximes) can
reactivate the enzyme much more rapidly than sponta-
neous hydrolysis. Wilson and co-workers accomplished
the reactivation by use of the active-site-specific nu-
cleophile pralidoxime. This compound, oriented by its
quaternary nitrogen atom, brings about a nucleophilic at-
tack on the phosphorus atom, leading to the formation of
an oxime-phosphonate-enzyme complex that dissociates
into oxime-phosphonate and free enzyme (Figure 6-10).
Pralidoxime has found use in the treatment of organophos-
phorous poisoning and is most active in relieving the inhi-
bition of skeletal muscle acetylcholinesterase. The phos-
phorylated enzyme can also lose an isopropoxy residue;
such an enzyme-inhibitor complex has been named an
“aged” enzyme. The aged enzyme is resistant to regen-
eration by pralidoxime because the phosphorus atom is
no longer an effective center for nucleophilic attack. The
severity and duration of toxicity of organophosphorous
compounds depend on their lipid solubility, the stability of
the bond linking the phosphorus atom to the oxygen of the
serine hydroxyl group, and the ease with which the aged
enzyme complex is formed. Some organophosphorous
compounds are so toxic that they can be fatal within a few
seconds of exposure. Other manifestations (e.g., excessive
mucus secretion and bronchoconstriction) are reversed by
administration of atropine, which does not relieve en-
zyme inactivation but, by binding to some of the acetyl-
choline receptor sites, renders ineffective the accumulated
Organophosphorous compounds have been used in the
identification of functional groups essential for catalysis.
Another approach to identification of functional amino
acid residues is
affinity labeling.
The labeling is produced
by a synthetic substrate-like reagent designed to form a
covalent linkage with some amino acid residue at or near
the active site of the enzyme. After the labeling, the en-
zyme is subjected to classic techniques of degradative pro-
tein chemistry (Chapter 3) to determine the amino acid
sequence of the structurally altered portion of the enzyme,
which is inferred to be the active site.
Inactivation and Reactivation of
Cytochrome Oxidase
Cyanides are among the most rapidly acting toxic sub-
stances. Cyanide (CN- ) inhibits cellular respiration to
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