JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 21, S13–S14 (2001)DOI:10.1002/jat.790

Peripheral Site Ligands Accelerate Inhibitionof Acetylcholinesterase by NeutralOrganophosphates

Zoran Radic* and Palmer TaylorDepartment of Pharmacology, University of California at San Diego, La Jolla, CA 92093-0636, USA

Key words: acetylcholinesterase; acceleration; acylation; allosteric; organophosphorus compounds; peripheral ligands;peripheral site.

The rates of inhibition of mouse acetylcholinesterase (AChE; EC 3.1.1.7) by paraoxon, haloxon, DDVP andenantiomers of neutral alkyl methylphosphonyl thioates and cationic alkyl methylphosphonyl thiocholineswere measured in the presence and absence of AChE peripheral site inhibitors: gallamine, d-tubocurarine,propidium, atropine and derivatives of coumarin. All ligands, except the coumarins, at submillimolarconcentrations enhanced the rates of inhibition by neutral organophosphates, whereas inhibition rates bycationic organophosphates were decreased. When peripheral site ligand concentrations extended to millimolarconcentrations the extent of the enhancement decreased, creating a well-shaped activation profile. Analysisof inhibition by DDVP revealed that peripheral site inhibitors increase the second-order reaction rates byincreasing maximal rates of phosphorylation.

These observations suggest that peripheral site ligands are capable of allosterically affecting the confor-mation of residues in the choline binding site of AChE, thus optimizing the position of the leaving group ofuncharged organophosphates during the inhibition reaction. Copyright 2001 John Wiley & Sons, Ltd.

INTRODUCTION

Some inhibitors of acetylcholinesterase (AChE; EC3.1.1.7)-catalysed acetylthiocholine hydrolysis are knownto accelerate hydrolysis of certain neutral substrates. Theenzymic hydrolysis of a series of small neutral acetatesis accelerated, for example, in the presence of N-acetylacridinium cation.1 Similarly, binding of small cationicligands, such as tetramethyl-or tetraethyl ammonium, intothe AChE active center was shown to increase rates ofAChE acetylation by carbamyl and sulfonyl fluorides.2,3

The small size of these neutral acylating agents and thecorresponding cations that modulate the kinetics allow fortheir simultaneous coexistence within the AChE activecenter gorge.

In this study we have examined the acceleration ofthe rate of phosphylation of mouse AChE with neu-tral organophosphates but when accelerating ligands werebound to the peripheral site of AChE.

RESULTS AND DISCUSSION

Rate constants of inhibition of mouse AChE byparaoxon, DDVP, haloxon and enantiomers of neutralalkyl methylphosphonyl thioates were found to increase

* Correspondence to: Z. Radic, Department of Pharmacology, Univer-sity of California at San Diego, La Jolla, CA 92093-0636, USA.E-mail: zradic@ucsd.edu

in the presence of AChE peripheral site inhibitorsgallamine. d-tubocurarine, propidium and atropine(Fig. 1), but derivatives of coumarin reduced the rates ofphosphylation.

The same d-tubocurarine or gallamine concentrationsthat accelerated paraoxon inhibition reduced the ratesof hydrolysis of neutral (p-nitrophenyl acetate) and

Activator (mM)

AtropineGallamine

Propidium

0.00

0

2

4

6

10

8

12

14

0.01 0.1 1 10 100 1000

d-Tubocurarine

k A /

k

Figure 1. Relative rate constants (kA/k) of AChE inhibition byparaoxon in the presence of varying concentrations of fourperipheral site ligands. Constants are expressed relative to therate constants in the absence of any activator. Modified fromRef. 5.

Copyright 2001 John Wiley & Sons, Ltd.Received 27 March 1999

Accepted 10 April 2000

S14 Z. RADIC AND P. TAYLOR

00

20

40

60

80

100

120A

5000

DDVP (µM)

k (m

in−1

)

No Gallamine

+ 0.5 mM Gallamine

10000

B

DD

VP

/ k

(M *

min

)

No Gallamine

+ 0.5 mM Gallamine

00

20

40

60

80

100

120

140

5000

Ki = 4000 µMk2 = 167 min−1

Ki = 4600 µMk2 = 99 min−1

−5000

DDVP (µM)10000

Figure 2. Inhibition of AChE by DDVP as a function of its concentration and in the presence of gallamine: (A) plot of dependence of thefirst-order rate inhibition constants on DDVP concentration; (B) linearized form of the plot in (A). Intercepts of lines on abscissa in thispanel yield values of Ki, whereas their slopes yield values of k2, for inhibition of AChE by DDVP. Modified from Ref. 5.

charged (acetylthiocholine) substrates. Both activationof phosphylation and inhibition of substrate hydrolysisoccurred in a biphasic manner, consistent with the forma-tion of at least two different enzyme–inhibitor/acceleratorcomplexes. Formation of the accelerating complex wasshown primarily to increase the maximal rates of phos-phylation by DDVP (Fig. 2) while not affecting the DDVPaffinity for AChE.

The magnitude of enhancement of the inhibition rateswas reduced by amino acid replacements at the peripheralsite, whereas selective replacements at the choline bindingsite increased the magnitude of enhancement of inhibitionachieved with peripheral site ligands. The enhancementwas potentiated further by simultaneous substitution of

residues in the choline binding site and in the acyl pocketof the enzyme.

In summary, the acceleration of AChE acylation byneutral organophosphates can be interpreted in termsof binding of the accelerating ligand to an allostericsite. Binding of a peripheral site ligand on the surfaceof the enzyme enhances the rate of acylation occur-ring in its active center. Presumably this occurs byinfluencing a change in conformation, therein the ori-entation of the leaving group of the organophosphate.Thus, as suggested in previous studies,4 the periph-eral site at the surface of the enzyme is linked tothe active center through a conformational change ofprotein.

REFERENCES

1. Barnett P, Rosenberry TL. Catalysis by acetylcholinesterase.Acceleration of the hydrolysis of neutral acetic acid estersby certain aromatic cations. J. Biol. Chem. 1977; 252:7200–7206.

2. Metzger HP, Wilson IB. Acceleration of the rate of reactionof dimethylcarbamyl fluoride and acetylcholinesterase bysubstituted ammonium ions. J. Biol. Chem. 1963; 238:3432–3435.

3. Kitz R, Wilson IB. Acceleration of the rate of reactionof methanesulfonyl fluoride and acetylcholinesterase by

substituted ammonium ions. J. Biol. Chem. 1963; 238:745–748.

4. Berman HA, Taylor P. Fluorescent phosphonate label forserine hydrolases. Pyrenebutyl methylphosphonofluoridate:reaction with acetylcholinesterase. Biochemistry 1978; 17:1704–1713.

5. Radic Z, Taylor P. The influence of peripheral site ligandsand chiral organophosphates with wildtype and mutantacetylcholinesterases. Chem. Biol. Interact. 1999; 119–120,111–117.

Copyright 2001 John Wiley & Sons, Ltd. J. Appl. Toxicol. 21, S13–S14 (2001)