Molecular Mechanisms for Hydrolytic Enzyme Action. III. A General Mechanism for the Inhibition of Acetylcholinesterase

Krupka, Richard M.; Laidler, Keith J.

doi:10.1021/ja01467a043

Cited by 42 publications

(39 citation statements)

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“…Reaction schemes involving cycles lead to exceedingly complicated rate equations which are not useful for the analysis of experimental data (23,24 [26] and [27] are in a particularly useful form for arriving a t the relationships that apply in special cases, and in deriving the conditions that apply to systems that have been studied experimentally. Two extreme cases are represented by kz << ka (acylation rate-determining) and kg << kz (deacylation rate-determining).…”

Section: The General Inhibition Equationsmentioning

confidence: 99%

The influence of pH and inhibitors on the kinetics of enzyme reactions involving two intermediates

Kaplan¹,

Laidler²

1967

Can. J. Chem.

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General steady-state equations are worked out for enzyme reactions which occur according to the scheme E + S = E S 3 E S ' -E + I ' z . PIEquations showing the pH dependence of the kinetic parameters are developed in a form which distinguishes between essential and nonessential ionizing groups. The p K dependence of i,/Zm, the second-order constant extrapolated t o zero substrate constant, gives pK values for groups which ionize on the free enzyme, but reveals such a p K only if the corresponding group is also involved in the breakdown of the Michaelis complex. General steady-state equations are also developed for the case in which a n inhibitor can combine with the free enzyme, the enzyme-substrate complex, and also a second intermediate (e.g. a n acyl enzyme). The equations are given in a form that is convenient for analyzing the experimental results, and a number of special cases are considered. I t is shown how the type of inhibition depends not only on the natureof the inhibitor but also on that of the substrate, a n important factor being the rate-determining step of the reaction. Examples of the various kinds of behavior are given.

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Section: The General Inhibition Equationsmentioning

confidence: 99%

The influence of pH and inhibitors on the kinetics of enzyme reactions involving two intermediates

Kaplan¹,

Laidler²

1967

Can. J. Chem.

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“…Binding of a second substrate molecule at a peripheral site could affect active-center conformation to reduce acylation rates. Alternatively, binding of a second substrate molecule to the acyl-enzyme may selectively influence deacylation (42).…”

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confidence: 99%

Mutagenesis of essential functional residues in acetylcholinesterase.

Gibney

Camp

Dionne

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

131

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The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignnment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence ofa catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrateinhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.Efficiently catalyzed hydrolysis of the neurotransmitter acetylcholine is critical to proper functioning of the cholinergic nervous system, and several pharmacologic and toxicologic agents act by inhibiting acetylcholinesterase (AChE; acetylcholine acetylhydrolase, EC 3.1.1.7). AChE functions as a serine hydrolase (1-3) and enzyme inhibitors used clinically or as insecticides typically serve as alternative substrates by acylating the active-center serine with slower leaving groups. The primary structure of AChE revealed that it lacks global sequence similarities with serine hydrolases of the trypsin and subtilisin families and only possesses residue identity with trypsin immediately around the active-center serine (4). Surprisingly, AChE is homologous to the carboxyl-terminal domain of a large secreted glycoprotein, thyroglobulin, the precursor to thyroid hormone (4). Subsequent primary structures showed that the cholinesterases belong to a distinct, but functionally eclectic, family of serine hydrolases. Included in this family are the butyrylcholinesterases, hepatic microsomal carboxylesterases, the Drosophila Est-6, lysophospholipases, cholesterol esterases, and two proteins found in inclusion bodies of Dictyostelium (5-11).The cholinesterases are characterized by their specificity for choline esters but can be subdivided on the basis of acyl group selectivity. The true AChEs (EC 3.1.1.7) show weak catalytic activity for choline esters with acyl groups larger than propionylcholine; the butyrylcholinesterases (EC 3.1.1.8) efficiently hydrolyze esters with large...

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“…This enzyme is totally inhibited in the electroplax preparation, and there are differences between the two phenomena. The inhibition of acetylcholinesterase is revealed in the catalytic rather than the binding step and is due to the blockade of deacetylation by high ACh concentrations (7). Also, inhibition of the enzyme occurs at concentrations higher than 1 mM rather than at micromolar concentrations of ACh.…”

Section: Discussionmentioning

confidence: 99%

Autoinhibition of Acetylcholine Binding to Torpedo Electroplax; A Possible Molecular Mechanism for Desensitization

Eldefrawi

O’Brien

1971

Proc. Natl. Acad. Sci. U.S.A.

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Molecular Mechanisms for Hydrolytic Enzyme Action. III. A General Mechanism for the Inhibition of Acetylcholinesterase

Cited by 42 publications

References 0 publications

The influence of pH and inhibitors on the kinetics of enzyme reactions involving two intermediates

The influence of pH and inhibitors on the kinetics of enzyme reactions involving two intermediates

Mutagenesis of essential functional residues in acetylcholinesterase.

Autoinhibition of Acetylcholine Binding to Torpedo Electroplax; A Possible Molecular Mechanism for Desensitization

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