Decarbamoylation of acetylcholinesterases is markedly slowed as carbamoyl groups increase in size.

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. We show here, in contrast to a previous report, that decarbamoylation rate constants are independent of the leaving group for a series of carbamates with the same carbamoyl group. When the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-dimethyl-, N-ethyl-N-methyl-, or N,N-diethyl-, the decarbamoylation rate constants decreased by 4-, 70-, and 800-fold, respectively. We suggest that this relationship arises as a result of active site distortion, particularly in the acyl pocket of the active site. Furthermore, solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site.

Interactions between the peripheral site and the acylation site in acetylcholinesterase.

Acetylcholinesterase (AChE) hydrolyzes its physiological substrate acetylcholine at one of the highest known catalytic rates. Two sites of ligand interaction have been identified: an acylation site or A-site at the base of the active site gorge, and a peripheral site or P-site at its mouth. Despite a wealth of information about the AChE structure and the role of specific residues in catalysis, an understanding of the catalytic mechanism and the role of the P-site has lagged far behind. In recent years we have clarified how the P- and A-sites interact to promote catalysis. Our studies have revealed that the P-site mediates substrate trapping and that ligand binding to the P-site can result in steric blockade of the A-site as well as allosteric activation. We have demonstrated this activation only for the acylation step of the catalytic reaction, but others have proposed that it involves the deacylation step. To investigate this point, we have measured the reaction of carbamoyl esters (carbamates) with AChE. With these slowly hydrolyzed substrates, the carbamoylation (acylation) and decarbamoylation (deacylation) steps can be resolved and analyzed separately. Carbamoylcholine is one of the closest structural analogs of acetylcholine, and we monitored these steps in continuous mixed assays with acetylthiocholine as a reporter substrate. At high concentrations of carbamoylcholine, decarbamoylation was inhibited but no activation of carbamoylation was observed. However, high concentrations of acetylthiocholine had no effect on the decarbamoylation rate constants. We concluded that the binding of acetylthiocholine to the P-site does not activate deacylation reactions.