The inhibition of acetylcholinesterase by arsenite and fluoride

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 226, No. 2, October 15, pp. 492-497, 1933

The Inhibition of Acetylcholinesterase JIMMY Depwtment

of Chemistry,

D. PAGE’ University

by Arsenite and Fluoride’

AND I. B. WILSON of Colurado,

Boulder,

Colorado

80309

Received March 3, 1933, and in revised form June 29, 1983

The effect of fluoride on the rate of reaction of acetylcholinesterase with arsenite, on the rate of dissociation of the enzyme-arsenite complex, and on the equilibrium between enzyme and arsenite was studied. Fluoride decreases the rate of the reaction between acetylcholinesterase and arsenite and changes the apparent equilibrium dissociation constant between the enzyme and arsenite, but even at concentrations as high as 0.2 M has no effect on the rate of dissociation of the enzyme-arsenite complex. The binding of fluoride and arsenite with the enzyme is highly anticooperative and may well be mutually exclusive. These results are consistent with a model in which the binding sites overlap and in which the same functional groups are involved. The inhibition of acetylcholinesterase (acetylcholine hydrolase EC 3.1.1.7; AChE)3 by arsenite and fluoride are puzzling phenomena because AChE does not contain the structural features usually associated with the inhibition of enzymes by these agents. Trivalent arsenic compounds are potent inhibitors of a number of enzymes (1) but the mechanism of this inhibition is the reaction of the arsenical with free sulfhydryl groups, notably those of reduced lipoic acid, to form cyclic thio-arsenite diesters (2). AChE, however, is not inhibited by other compounds known to be sulfhydryl reagents such as organomercury compounds or iodoacetamide (3). The enzyme has been found to contain cysteine only in the form of disulfide bridges and not as free thiol (4). Arsenite inhibits AChE in a second-order reversible reaction with a dissociation constant of about lop5 M which is comparable to the value for arsenite and lipoic

acid containing enzymes. The inhibition rate constant for arsenite with AChE is small, -lo2 M-l min-‘. Arsenite can be considered a quasi-irreversible inhibitor because dissociation is slow, -10e3 min-‘. The rate constants are highly pH dependent, increasing markedly above pH 7.5 (5). Although arsenite exists primarily as undissociated arsenious acid below pH 9, we have followed customary usage in referring to the compound as arsenite for all pH values. Fluoride reacts rapidly and reversibly with AChE (6, 7). It is less potent than arsenite and has a dissociation constant on the order of 5 X 10V4 at neutral pH. However, the constant is markedly dependent on pH and increases rapidly in alkaline solutions. The nature of the complex between AChE and fluoride is not known. Other enzymes inhibited by fluoride require a metal ion for activity, and fluoride inhibition is due to fluoride entering the coordination sphere of the required metal ion (8,9). But AChE neither contains metal ions nor needs metal ions for activity. We therefore need to look for other kinds of binding modes for fluoride to account for its inhibition of AChE. It appears that both fluoride and arsenite

’ This work has been supported by U.S.P.H.S. Grant ROl NS7156-16. * To whom all correspondence should be addressed. 8 Abbreviations used: AChE, acetylcholinesteraae; DTNB, 5,5’-dithiobis-(2-nitrobenzoic acid): AThChI, acetylthiocholine iodide. 0003-9861/83 $3.00 Copyright All rights

0 1983 by Academic Press. Inc. of reproduction in any form reserved.

492

INHIBITION

OF ACETYLCHOLINESTERASE

react near the active site of AChE since reversible competitive inhibitors of the enzyme are antagonists toward the binding of fluoride and arsenite (5, 6). In order to determine whether these two inhibitors bind to the same site and funetional groups of the enzyme, we have studied the effect of fluoride on (i) the rate of association of arsenite with the enzyme, (ii) the rate of dissociation of the enzymearsenite complex, and (iii) the equilibrium between arsenite and the enzyme. MATERIALS

AND METHODS

Materials Glyclyglycine-HCL, 5,5’-dithio-bis-2-nitrobenzoic acid (DTNB), acetylthiocholine iodide (AThChI), phenylarsenoxide, and boric acid were obtained from Sigma. Phenylhydrazine was purchased from Matheson, Coleman and Bell. Sodium fluoride was from J. T. Baker Chemical Company, sodium arsenite was from Fisher Scientific, and paminophenyl arsenic acid (parsanilic acid) was from Aldrich. Enzyme. The enzyme used in these experiments was the 11 S form from the electric organ of Electrophorus electricus obtained from Sigma and purified by affinity chromatography (10) to a specific activity of 6200 units/mg and a concentration of 1200 units/ ml. No significant kinetic differences were observed between purified and unpurified enzyme. AChE assays. AChE was assayed using a modified Ellman method (11). The cuvette contained 3.0 ml of 0.05 M phosphate buffer, pH 7.4,0.4 M NaCl, 3 X lo-’ M DTNB and 7.5 X lo-’ M AThChI. Enzyme solution (usually 0.01 units in 10 ~1) was then added and the absorbance change at 412 nm was monitored using a Varian Model 635 uv-visible spectrophotometer, thermostated at 25°C in conjunction with a Varian A-25 recorder. InhibitimL rate w&ants (k). Enzyme samples were diluted to the desired activity (-1 unit/ml) in a buffer of the desired composition, i.e., fluoride concentration, pH, and assayed for 100% activity. At time zero the desired amount of arsenite was injected into the enzyme solution by syringe and mixed, and an aliquot was assayed at various times. The resulting activities were plotted as percentages of the original activity on a semi-log plot. The tljz and b were then determined both graphically and from a linear regression of the data points. Llissociatiun rate castants (kJ. A 2 X 10m6enzyme solution was inhibited to approximately 10% activity with arsenite (-1 X lo-’ M). At time zero an aliquot of inhibited enzyme was diluted 1000X into a solution of the desired composition and aliquots were assayed at various times. Uninhibited enzyme was treated and assayed in the same way except that no arsenite

BY ARSENITE

AND FLUORIDE

493

was added. Percentage inhibition vs time was then plotted on a semi-log plot and the tllz and k, were determined both graphically and by linear regression of the data points. Equilibrium constants for arsenite inhibition (Kd and &). Enzyme was diluted into solutions of varying fluoride and arsenite concentrations at pH 8.5. Controls were also diluted into buffers of the same composition but lacking arsenite. Controls were used to determine 100% activity and as a check on the stability of the enzyme for the time required to reach equilibrium. The inhibited enzyme solutions were allowed to equilibrate (determined by lack of change in the percentage inhibition) and multiple assays were done for each fluoride and arsenite concentration. The dissociation constant Kd or apparent dissociation constant Kd was then calculated from f.1 Kd = (1 - f) ’ where f is the fraction of enzyme activity. Synth.esis of paminophenylarserwxide The p-arsanilic acid was recrystallized from hot water and reduced with phenylhydrazine in methanol (12). RESULTS

The definition and measurement of various constants are based upon the schematic representation of the reactions. Our equations allow for the possible formation of a ternary complex of enzyme (E), arsenite (I) and fluoride (F), although, as we shall see, our measurements suggest that this complex probably cannot form and that the binding of arsenite and fluoride is highly anticooperative and possibly mutually exclusive. The scheme appropriate for the effect of fluoride on the dissociation constant of the enzyme-arsenite complex is F+E+I---

II -:IF E.F+IB

F+E.I

[I E-1-F

where, as indicated, all constants are dissociation constants. The apparent enzymearsenite dissociation constant that we measure is defined as

PAGE

AND

where f is the fractional enzyme activity. Although E. I does not dissociate in the assay, fluoride dissociates completely from the enzyme so that, along with E, Es F is measured as active enzyme. The complexes E -1 and E -1-F are inactive complexes. From the scheme we have Rh=Kg;;z). This equation

WILSON

s f

can be put in the linear form

1

1 -Kd/Kd 1 =l-KF/Kjj+l-KF/K”F*

KF

I

PI

F’

Our results, plotted in accordance with this equation in Fig. 1, show that the intercept does not differ perceptibly from 1.00. Linear regression gives an intercept value of 1.002 and a slope of 8.8 X 1O-3 M. This value of the intercept shows that K$ is very much larger than KF and may well be infinite. Under this circumstance, the slope (8.8 X 10P3 M) is KF. An infinite V&W for FF means that the ternary complex does not occur and the binding of arsenite and fluoride are mutually exclusive.

FIG. 2. Inhibition of AChE by arsenite in varying fluoride concentrations. Inhibition was done with 10m3 M arsenite at pH 7.4 in a buffer of 50 mM NaHzPO,, 0.4 M NaCl + NaF. Enzyme concentration was 2 X lo-* M. 0, No F-; 0, 5 X lo+ M F-; 0, lo+ M F-; A, 0.05 M F-.

(The four constants

are not independent;

I(*F/KF = K$'/KJ. The scheme for the effect of fluoride on the rate of inhibition of the enzyme by arsenite is k E+I-E.1 ski E-F + I+ E-1-F. This yields the pseudo-first-order log;

equation

= -k:.I.t, 0

6.0

where the pseudo-first-order

constant

is

5.0. 40. 1 ___ l-(K,,/K&

30

100

200

300

400

500

?&-I

FIG. 1. Plot of apparent equilibrium data according to Eq. [l]. Equilibria were measured at pH 8.5 in a buffer of 20 mM glycylglycine-HCl, 0.21’7 Y NaCl + NaF. Equilibria were reached overnight. Enzyme concentration was 2 X lOTa M in all cases. Arsenite concentration was varied from 1.0 X 1O-6 to 2.0 X lo-’ M in order to get between 40 and 60% enzyme activity. Linear regression of the data points gives an intercept of 1.002 and a slope of 8.81 X lo-‘.

Figure 2, for pH 7.4, shows that the pseudofirst-order rate is obeyed and that fluoride slows the rate of reaction of arsenite with the enzyme. Since the rate is very slow at 0.05 M F, it is apparent that (Yis very small. The above equation can be put in the linear form 1 1 - k;/ki

1 KF = 1--Ly + l--(y

1 - F.

PI

Our results, plotted for pH 8.5 in accordance with this equation in Fig. 3, show that the intercept does not differ perceptibly from 1.00. Linear regression gives an intercept of 1.003 and a slope of 5.2 X lo-” M. The value of the intercept indicates that

INHIBITION

OF

ACETYLCHOLINESTERASE

1.6.

14. 1 1 -(k;/k,)

20

40

60

SO

100

'/F-l

FIG. 3. Plot of inhibition rate constant data according to Eq. [Z]. Rates were determined at pH 8.5 in a buffer containing 20 mEd glycylglycine-HCl, 0.217 M NaCl + NaF. Enzyme concentration was 2 X lo-* M and arsenite concentration was 1.1 X lo-’ M. High concentrations of fluoride were used to emphasize the intercept. Linear regression of the data points gives an intercept of 1.003 and a slope of 5.19 X 10d3.

(Yis very much smaller than 1.0 and may well be zero. Finally, the scheme for the effect of fluoride on the dissociation of the arseniteenzyme complex,

BY

ARSENITE

AND

FLUORIDE

495

We looked into the question of whether the reaction between arsenite and the enzyme might involve the formation of a reversible complex prior to the formation of the quasi-irreversible complex by measuring rates of inactivation of the enzyme at high arsenite concentrations up to 0.05 M. There was no evidence of a reversible complex; second-order kinetics were followed throughout the inhibitor range. Because borate is far more effective than arsenite in forming cyclic diesters with diols, we studied the effect of borate on the enzyme. Borate does not affect the activity of the enzyme nor does it affect the rate of its inactivation by arsenite nor the equilibrium between arsenite and AChE. We did a few experiments with phenylarsenoxide and p-aminophenylarsenoxide. Although they are less potent than arsenite (Kd = 2 X 10M3 M), they inhibit the enzyme slowly (56 M-’ min-‘) and reversibly. Similarly to arsenite, association and dissociation are both accelerated by pyridine-

E.I2E+I E+FTE.F+I, yields the first-order

equation

E,, - E = -k$, 1% ___E 0

where k:=k,(;:y;z). The above equation 1 1 - k:/k,

can be linearized 1

K*

to

1

=~+l-F/j*$

In our experiments at pH 8.5, where the rate of dissociation is much more rapid than at pH 7.0, we found no effect of fluoride on the rate of dissociation of the enzyme-arsenite complex, so that k: = k, (Fig. 4). This result indicates that either /3 = 1 or K$ is infinite. The effect of fluoride on the apparent dissociation constant of the enzyme-arsenite complex had already shown that I(*F was very large and probably infinite.

Min FIG. 4. Recovery of AChE activity after inhibition by arsenite and then dilution of the arsenite. Determinations of recovery rates were done in 20 mM glycylglycine-HCl, 0.217 M NaCl + NaF. Stock solution of enzyme was inhibited with lo-’ M arsenite and diluted 1000X into the pH 8.5 buffer. Final enzyme concentration was 2 X 10-O M. 0, No F-; A, 0.217 M F-.

496

PAGE

AND

2-aldoximine methiodide. Fluoride slows inhibition and changes the apparent equilibrium constant but it does not affect the dissociation rate (0.11 min-‘). The binding of fluoride to AChE as a function of pH was obtained from the effect of fluoride on the rate of the reaction of arsenite with the enzyme (Table I). DISCUSSION

The effect of fluoride on the reaction of arsenite with AChE in which the reaction with arsenite is prevented but the dissociation of arsenite is unaffected resembles the effect of fluoride on the reaction of carbamates and methane-sulfonates with this enzyme (13,14). The latter two inhibitors react with the nucleophilic serine hydroxyl group of AChE. This suggests that the active site serine might be one of the ligands binding arsenite and fluoride. Even if this is so, there might be other groups involved, since neither arsenite nor fluoride inhibit other serine esterases or proteases. Although arsenite does form cyclic diesters with polyols such as mannose and cathecho1 (15), it is unlikely that formation of a diester is involved in the inhibition of AChE since borate, which forms much more stable diesters than arsenite, neither TABLE pH DEPENDENCE PH

5.5 6.0 6.5

I

OF F- BINDING

KF

TO AChE

(mW

0.068 0.11 0.22

8.0

0.50 1.4 2.6

8.5

8.7

7.0

7.5

9.0

20

Note, KFs were determined by plots of Eq. [2] from data at the various pHa. Inhibition rate constants (Q were determined in 20 mM glycylglycine-HCI or NarHPO, buffers with 1 = 0.250. The arsenite concentration used was 1.1 X lo-’ M and the enzyme concentration was 2 X lo-* M in all cases.

WILSON

inhibits the enzyme nor blocks the inhibition by arsenite. Our measurements show that fluoride and arsenite are probably mutually exclusive. The simplest interpretation of this antagonism is that they bind to at least one enzyme structure in common, particularly since they both seem to bind very near the active site. Both arsenite and fluoride have limited possibilities for binding due to their simple structures. It is possible to propose a model for the binding of these two inhibitors that accounts for our observations and is based upon the properties of fluoride and arsenite. Fluoride can form ionic and hydrogen bonds, and, in ammonium fluoride crystals, fluoride forms four hydrogen bonds with tetrahedrally arranged ammonium ions (16). Possible “ammonium” ions in a protein would be contributed by lysine, arginine, and histidine. Fluoride can also form hydrogen bonds with the hydroxyl groups of serine, threonine, or tyrosine. Arsenite can also form hydrogen bonds with these same groups; it can also form esters. We can therefore envision a model in which fluoride forms hydrogen bonds with one hydroxyl group and one or more “ammonium ions.” Arsenite forms an ester with the same hydroxyl group and also forms hydrogen bonds. Thus the binding of fluoride and arsenite would be mutually exclusive (or at least highly antagonistic). The formation of an ester would explain the slow inhibition by arsenite and the slow recovery of enzyme activity on dilution. The increased binding of fluoride as the pH is decreased probably is caused by an increase in electrical charge; arsenite, which is not charged below pH 9, does not bind better as the pH is lowered. REFERENCES 1. BOYER, P. D., LARDY, H., AND MYRBACK, K., eds. (1963) The Enzymes, 2nd ed., Academic Press, New York. 2. PETER, R. A. (1949) Sgmp. Sot Exp. Biol 3, 3659. 3. MOUNTER, L. A., AND WHITTAKER, V. P. (1953) Biocbm. .I 53, 16'7-1'73. 4. ROSENBERRY, T. L. (1975) A&an. Enqmol43,103-

218.

INHIBITION

OF

ACETYLCHOLINESTERASE

5. WILSON, I. B., AND SILMAN, I. (1977) Biochemistry 16,2’701-2’708. 6. KRUPKA, R. M. (1966) Mel PhurmucoL 2,558-569. 7. HEILBRONN, E. (1965) Actu Chem Scunu! 19.1333. 8. MACFARLANE, M. G., AND KNIGHT, B. C. J. G. (1942) Biochem. J. 35,884. 9. KEILIN, D., AND HARTREE, E. F. (1951) Biochem. J. 49, 88. 10. ASHANI, Y., AND WILSON, I. B. (1972) B&him. Bie phys. Acta 276, 228-233. 11. ELLMAN, G. L., COURTNEY, K. D., ANDRE% V., JR., AND FEATHERSTONE, R. M. (1961) Biochem Pharmaco~ 7, 88-95.

BY

ARSENITE

12. EHRLICH, Deb%.

AND

497

FLUORIDE

P., AND BERTHEIM, A. (1910) Chemi GeselL 43,917-927.

Be-richte

d

13. GREENSPAN, Pharmacol

C. M., AND WILSON, 6,266-272.

I. B. (1970)

Mol.

14. GREENSPAN, Pharmacol.

C. M., AND WILSON, 6,460-467.

I. B. (1970)

Mol.

15. ROY, G. L., LAFERRIERE, A. L., AND EDWARDS, J. 0. (1957) J. Iwrg. Nucl. Chem. 4, 106-114. 16. PAULING, L. (1940) The Nature Bond, 2nd ed., p. 288, Cornell Ithaca, New York.

of the Chemical University Press,