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

Abstract

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.

Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition

“…Among the 111 compounds identified from the primary HTS, 60 employ at least one reactive functionality that can form a covalent bond with the Serine residue (Ser203) in the AChE active site ( Fukuto 1990 ; Johnson et al. 2011 ; Venkatasubban et al. 2018 ).…”

“…Within this group, we identified one alkylated pyridine ( Johnson et al. 2011 ), 37 OPs ( Fukuto 1990 ), and 22 carbamates ( Venkatasubban et al. 2018 ).…”

“…2008 ). This hydrolysis process is also influenced by the size of the carbamoyl groups ( Venkatasubban et al. 2018 ).…”

Solvent Deuterium Oxide Isotope Effects on the Reactions of Organophosphorylated Acetylcholinesterase

“…In Table 1 , the k H for diethylphosphorylated AChE is about 75-fold lower than that for dimethylphosphorylated AChE. A similar trend was seen with carbamoylated AChE, where k 3 for N,N -diethylcarbamoyl AChE was about 300-fold lower than that for N , N -dimethylcarbamoyl AChE [ 2 ]. The difference for the organophosphorylated AChEs may result from distortion of the AChE active site by the larger diethylphosphoryl group as seen in Figure 2 A.…”

“…This complex is an intermediate with acetylthiocholine but is not detectable, as explained in Equation (1) below. While the hydrolysis of the carbamoyl or organophosphoryl intermediates involves transfer of their acyl group from serine to water, their deacylation rate constants ( k 3 or k H ) are orders of magnitude smaller than that of acetylated AChE [ 2 , 3 ]. Their slow deacylation makes them potent inhibitors of AChE, and some carbamates and organophosphates (also denoted OPs) have been widely used as insecticides [ 4 ].…”