Catalysis by Acetylcholinesterase. The Rate-Limiting Steps Involved in the Acylation of Acetylcholinesterase by Acetic Acid Esters and Phosphorylating Agents

Inferences about the catalytic mechanism of acetylcholinesterase are frequently made on the basis of a presumed analogy with chymotrypsin. Although both enzymes are serine hydrolases, ;several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, k0a1/Kapp· While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysi·s, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of k0.JKapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves non-linear inhibition by H+. Deuterium oxide isotope effects of k0.JKapp range from 1.1 for acetylchoHne to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit ·step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate. Other neutral acetic acid esters give apparent pK3 values and deuterium oxide isotope effects consistent with rate-limiting general-base catalysis at all substrate concentrations. The pH dependence of second-order acylation by two phosphorylating agents was also examined; one of these agents, diisopropylfluorophosphate, gave the very unusual observation of an increase in acylation rate as the pH decreased from 6 to 5

Catalysis by Acetylcholinesterase. The Rate-Limiting Steps Involved in the Acylation of Acetylcholinesterase by Acetic Acid Esters and Phosphorylating Agents

Inferences about the catalytic mechanism of acetylcholinesterase are frequently made on the basis of a presumed analogy with chymotrypsin. Although both enzymes are serine hydrolases, ;several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, k0a1/Kapp· While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysi·s, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of k0.JKapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves non-linear inhibition by H+. Deuterium oxide isotope effects of k0.JKapp range from 1.1 for acetylchoHne to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit ·step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate. Other neutral acetic acid esters give apparent pK3 values and deuterium oxide isotope effects consistent with rate-limiting general-base catalysis at all substrate concentrations. The pH dependence of second-order acylation by two phosphorylating agents was also examined; one of these agents, diisopropylfluorophosphate, gave the very unusual observation of an increase in acylation rate as the pH decreased from 6 to 5

Interaction of ligands with acetylcholinesterase. Use of temperature-jump relaxation kinetics in the binding of specific fluorescent ligands

Rosenberry TL, Neumann E. Interaction of ligands with acetylcholinesterase. Use of temperature-jump relaxation kinetics in the binding of specific fluorescent ligands. Biochemistry. 1977;16(17):3870-3878

Solvent Deuterium Oxide Isotope Effects on the Reactions of Organophosphorylated Acetylcholinesterase

Organophosphates (OPs) are esters of substituted phosphates, phosphonates or phosphoramidates that react with acetylcholinesterase (AChE) by initially transferring the organophosphityl group to a serine residue in the enzyme active site, concomitant with loss of an alcohol or halide leaving group. With substituted phosphates, this transfer is followed by relatively slow hydrolysis of the organophosphoryl AChE, or dephosphorylation, that is often accompanied by an aging reaction that renders the enzyme irreversibly inactivated. Aging is a dealkylation that converts the phosphate triester to a diester. OPs are very effective AChE inhibitors and have been developed as insecticides and chemical warfare agents. We examined three reactions of two organophosphoryl AChEs, dimethyl- and diethylphosphorylated AChE, by comparing rate constants and solvent deuterium oxide isotope effects for hydrolysis, aging and oxime reactivation with pralidoxime (2-PAM). Our study was motivated (1) by a published x-ray crystal structure of diethylphosphorylated AChE, which showed severe distortion of the active site that was restored by the binding of pralidoxime, and (2) by published isotope effects for decarbamoylation that decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE. We previously reconciled these results by proposing a shift in the rate-limiting step from proton transfer for the small carbamoyl group to a likely conformational change in the distorted active site of the large carbamoyl enzyme. This proposal was tested but was not supported in this report. The smaller dimethylphosphoryl AChE and the larger diethylphosphoryl AChE gave similar isotope effects for both oxime reactivation and hydrolysis, and the isotope effect values of about two indicated that proton transfer was rate limiting for both reactions

Secondary Structure and Interfacial Aggregation of Amyloid-β(1â40) on Sodium Dodecyl Sulfate Micelles

Alzheimer\u27s disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that fibrillar Aβ and especially soluble Aβ aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Aβ aggregates generated from monomeric Aβ in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily β-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Aβ aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Aβ aggregation have found that amphiphilic interactions and micellar-like Aβ structures may play a role. We previously described the formation of Aβ(1−40) aggregates at polar−nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Aβ(1−40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Aβ(1−40) was introduced. With HFIP, the change involved an increase in predominant β-structure content and in fluorescence with thioflavin T, while with SDS, a partial α-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in β-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Aβ(1−40) monomers effectively

Effective charge on acetylcholinesterase active sites determined from the ionic strength dependence of association rate constants with cationic ligands

Nolte H-J, Rosenberry TL, Neumann E. Effective charge on acetylcholinesterase active sites determined from the ionic strength dependence of association rate constants with cationic ligands. Biochemistry. 1980;19(16):3705-3711