Info

Adapted from Heath, D. F.: Organophosphorus Poisons—Anticholinesterases and Related Compounds. New York, Pergamon Press, 1961.

aRelative rates at approximately optimal substrate concentration; rate with acetylcholine = 100.

Adapted from Heath, D. F.: Organophosphorus Poisons—Anticholinesterases and Related Compounds. New York, Pergamon Press, 1961.

aRelative rates at approximately optimal substrate concentration; rate with acetylcholine = 100.

form an acetyl-enzyme intermediate at a rate (k2) that is slower than either the association or dissociation rates. Choline is released from this complex with the formation of the acetyl-enzyme intermediate, EA. This intermediate is then hydrolyzed to regenerate the free enzyme and acetic acid. The acetylation rate, k2, is the slowest step in this sequence and is rate-limiting (see discussion that follow).

Kinetic studies with different substrates and inhibitors suggest that the active center of AChE consists of several major domains: an anionic site, to which the trimethylammonium group binds; an esteratic site, which causes hydrolysis of the ester portion of ACh; and hydrophobic sites, which bind aryl substrates, other uncharged ligands, and the alkyl portion of the acyl moiety of ACh. There is also a peripheral anionic site, removed by at least 20 A from this active center, which al-losterically regulates activity at the esteratic site.49 The anionic site was believed to have been formed by the y-carboxylate group of a glutamic acid residue,50 but more recent studies suggest that the aromatic moieties of tryptophan and pheny-lalanine residues bind the quaternary ammonium group of ACh in the anionic site through cation—^ interactions.51 The location and spatial organization in the esteratic site by serine, histidine, and glutamic acid residues constitute the esteratic site. The triad of these amino acid residues contributes to the high catalytic efficiency of AChE (Fig. 17.14).52

AChE attacks the ester substrate through a serine hy-droxyl, forming a covalent acyl-enzyme complex. The serine is activated as a nucleophile by the glutamic acid and histidine residues that serve as the proton sink to attack the carbonyl carbon of ACh. Choline is released, leaving the acetylated serine residue on the enzyme. The acetyl-enzyme intermediate is cleaved by a general base catalysis mechanism to regenerate the free enzyme. The rate of the deacety-lation step is indicated by k3.

Carbamates such as carbachol are also able to serve as substrates for AChE, forming a carbamylated enzyme intermediate (E-C). The rate of carbamylation (k2) is slower than the rate of acetylation. Hydrolysis (k3, decarbamylation) of the carbamyl-enzyme intermediate is 107 times slower than that of its acetyl counterpart. The slower hydrolysis rate limits the optimal functional capacity of AChE, allowing carbamate substrates to be semireversible inhibitors of AChE.

In the mechanism above, k3 is rate-limiting. The rate k2 depends not only on the nature of the alcohol moiety of the ester but also on the type of carbamyl ester. Esters of carbamic acid are better

carbamylating agents of AChE than the methylcarbamyl and dimethylcarbamyl analogs.53

O-C-rGlu-327

His-440

O-C-rGlu-327

His-440

His-440

Ser-200

His-440

Organophosphate esters of selected compounds can also esterify the serine residue in the active site of AChE. The hydrolysis rate (k3) of the phosphorylated serine is extremely slow, and hydrolysis to the free enzyme and phosphoric acid derivative is so limited that the inhibition is

TABLE 17.6 Inhibition Constants for Anticholinesterase Potency of Acetylcholinesterase Inhibitors

Reversible and Semireversible

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