STUDIES ON CHOLINESTERASE

VII. THE ACTIVE SURFACE OF ACETYLCHOLINE ESTERASE DERIVED FROM EFFECTS OF pH ON INHIBITORS*

BY IRWIN B. WILSON AND FELIX BERGMANNt

(From the Department of Neurology, College of Physicians and Surgeons, Columbia University, New York)

(Received for publication, February 10, 1950)

In previous investigations the kinetics of the inhibition of acetylcholine esterase were studied with two reversible inhibitors, prostigmine and eser- ine, and two irreversible inhibitors, diisopropyl fluorophosphate (DFP) and tetraethyl pyrophosphate (TEPP) (1, 2). One common feature resulted from the data obtained; namely, that all four inhibitors appear to act upon the same groups in acetylcholine esterase at which acetylcholine is hydrolyzed. The competition between inhibitor and substrate for the same surface region is suggested by two types of observations: (a) The sequence in which inhibitor and substrate come in contact with the en- zyme affects the course of hydrolysis in all cases. This suggests that the substrate has a protecting effect by competing with all four inhibitors for the active center. (h) When a concentrated enzyme solution is incubated with prostigmine prior to its incubation with DFP and later diluted, com- plete protection against DFP may be demonsbrated, suggest’ing that for- mation of the prostigmine-enzyme complex may occur at the same func- tional groups as those involved in the reaction of DFP with the enzyme.

In spite of this common feature, considerable difference exists between the various inhibit,ors. When, for example, the act,ivity-substrate con- centration relationship was tested in the presence of prostigmine, the opti- mum substrat,e concentration was progressively higher with increasing inhibitor concentration. This shift was also evident in the case of eserine, but smaller; nolie was observed with DFP and TEPP.

It appeared of interest to obtain more information about the factors affecting the mode of combination of the enzyme with the four inhibitors. Studies of the effects of pH changes are described in this paper.

Methods

The enzyme used throughout was highly purified acetylcholine esterase prepared from the electric tissue of Electrophorus electricus according to

*This investigation was supported by a grant from the Division of Research Grants and Fellowships of the National Institutes of Health, United States Public Health Service and from the Dazian Foundation for Medical Research.

t Present address, The Hebrew University, Jerusalem, Israel. 479

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480 CHOLINESTERbSF 2. VII

the method previously described (3). The hydrolysis of acetylcholine was determined in most cases with the calorimetric method (4), except in the dilution experiments wit*h TEPP, in which the manometric technique was applied. All experiments were carried out at room temperature (2O- 23”).

For pH 8.5 and below phosphate buffer of the following composition was used: 0.1 M Na2HP04, adjusted to the pH desired, 0.1 M NaCI, 0.0008 M MgCln, and 0.1 per cent gelatin. Above pH 8.5 the corresponding 0.1 M

borate buffer was used. Gelatin was employed to increase the stability of the highly diluted purified enzyme. The acetylcholine concentration was usually 3 to 4 X 1O-3 M final concentration, which is close to the op- timum.

The inhibitors used were prostigmine bromide, Hoffman-La Roche, eser- ine sulfate, Merck, and TEPP, Monsanto, of 40 per cent purity. Dimet*h- ylaminoethyl acetate hydrochloride (5) was recrystallized from a mixture of 1 part of ethanol in 20 pa& of ethyl acetat.e.’

Results

Prostigmine-Prostigmine (8.5 X lOA7 M) w&s incubated with acetyl- choline esterase for 20 minutes at pH values between 6 and 10. Acetyl- choline was then added to start the reaction and after 30 minutes samples were analyzed for the ester. The activity without prostigmine was deter- mined simultaneously. At pH levels above 8 controls mere run without the enzyme t,o correct for non-enzymatic hydrolysis. The correction is significant only above pH 9.

Prostigmine inhibits competitively. When acetylcholine is added after incubation of the enzyme with prostigmine, the substrate shows little com- petition with the inhibitor in the first few minutes. It takes about 20 to 30 minut,es until the new equilibrium is established. The data reported here therefore represent an average of the reaction before and after equi- librium.

The prostigmine cation remains unaltered with pH changes; the protein accepts or donates protons in conformity with the acidity of the medium. These changes in the protein molecule do not markedly affect the inhi- bition, as illustrated in Fig. 1.

.&e&e-Inhibition with eserine (3 X lo-’ M) was measured as with prostigmine. As with prostigmine, the competitive inhibition approaches equilibrium slowly; hence the data here reported are again an average of the period before and after equilibrium. Eserine differs from prostigmine in that it exists predominantly as a cation in neutral and acid solution and

1 We are greatly indebted to Dr. John A. Aeschlimann, Hoffmann-La Roche, Inc., for providing this compound and the prostigmine bromide.

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I. R. WILSON AND F. BERGMAIUX 481

as an uncharged base in alkaline medium. The relative amounts of cation and base at different pH are indicated in Fig. 2, which is a pH titration curve of the sulfate. The abscissa represents the volume of sodium hy- droxide but has been recalculated for the per cent of free base, since this is the more pertinent information. Therefore, the scale is not linear. Near the extremes of the curve the per cent of free base must be calculated from the ionization constant of the cation. The constant obtained from this curve, ~-ith the concentrations of base and cation but the activity of hydrogen ion, was 8 X 10e9 for an ionic strength of about 0.1. The decrease in inhibition at high pH, compared with the constant inhibition

z 80 0 I= m f 60

-I

I- = 40 0

3.:1L-- 6 7 8 9 IO

PH

FIG. 1. Inhibition of acetylcholine e&erase by prostigmine (0) and eserine (+) as a function of pH.

of prostigmine, as shown in Fig. 1, indicates that the cation of eserine is more potent as an inhibitor than is its uncharged base.

However, the base is also an inhibitor and the entire inhibition at pH 10.1 must be ascribed to this form (Table I). The dissociation constant for the eserine-enzyme complex is given by Augustinsson and Nichmansohn as 1.0 X lo-‘, from which the inhibition corresponding to the low cation concentration at pH 10 can be evaluated. The inhibition so determined is zero.

It may be noted that eserine was stable at pH 10 for the duration of t,he experiments (60 to 90 minutes). We found no significant decrease of inhibitory action after bringing the solution back to pH 7.0, in spite of the appearance of pink color in concentrated solution. Therefore, any influ- ence of chemical decomposition as a factor in the experiments at high pH may be excluded.

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482 CIIOLINESTERASE. VII

6

I I I I Ia, 0 24, 48 72 95Gi+99 PER CENT FREE BASE

FIG. 2. Titration curve of eserine. The original abscissa of ml. of NaOH was converted into the non-linear scale of free base.

TABLE I Inhibition of Acetylcholine Eeterase by Eserine (3 X 10-l a.r) As Function of pH in

Relation to Cation Present

PH I Per cent inhibition I Per cent cation (Zoo)

5.9 87,80 99 7 75,71 92 8 62 44 9 40 12

10.1 20 0.0

Dimeth.ylaminoethyl Acetate-Dimethylaminoethyl acetate (3.2 X 10e3 M) was studied as a substrate for acetylcholine esterase at pH levels between 6 and 10. This compound was estimated by the method employed for acetylcholine. At the optimum concentration of acetylcholine and at pH 7 this compound is hydrolyzed about 60 per cent aa fast as acetylcholine.

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I. B. WILSON ANB F. BERGMANN 433

From the titration curve of dimethylaminoethyl acetate the ionization constant was calculated in the same way as for eserine and found to be 5 x 10-S.

Acetylcholine is hydrolyzed enzymatically at a maximum rate between pH 7 and 9 (6). Since it exists only as cation, the effect of pH must be attributed exclusively to changes of the protein. The rate of hydrolysis of dimethylaminoethyl acetate as a function of pH is shown in Table II. In this case, however, the variations of the ratio of cation to base of the substrate also influence the rate of reaction. To evaluate this second factor separat,ely, the values observed must be corrected for the changing activity of the protein by comparison with the corresponding rates for acetylcholine hydrolysis. For example, the rate of hydrolysis of acetyl-

TABLE II

Hydrolysis of Dimethylaminoethyl Acetate (3.8 X lFa M) by Acetylcholine Esterase AI Function of pH

Calorimetric determination; 100 Klett units correspond to 1 PM of acetylcholine; temperature 20”.

PH Rate of hydrolysis

- -

6 7 8 9

10

-- men unifs

50

84

81 34 28

-

2

-

Rate relative to rcetylcholine (calculated)

mea units

84

91 81 34 34

-

_-

-

Per cent cation

98 92 75 16

1

choline at pH 6 is 60 per cent of the maximum. On the assumption that the protein changes in both cases quantitatively in the same way, the rate of hydrolysis of dimethylaminoethyl acetate at pH 6 may be divided by 0.6. The values so obtained are recorded in Table II.

Again the cation is the more active form, but the neutral molecule is also hydrolyzed, though at a much lower rate. At pH 10 the cationic concentration is 3.2 X 10m5 M. It may be inferred from acetylcholine that this concentration is too low to yield an appreciable rate of hydrolysis for the cationic form. Thus, the hydrolysis at pH 10 must be entirely due to the basic form.

TEPP-The influence of pH on the activity of TEPP was investigated by two types of experimental approach.

In one, concentrated solutions of acetylcholine esterase were incubated for 10 minutes with constant amounts of TEPP buffered at the pH re- quired. The mi.xture was then sufficiently diluted with gelatin-bicarbon-

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484 CHOLINESTERASE. VII

ate buffer to render the TEPP inactive. This method (2) is possible only with irreversible inhibitors. The activity of all solutions incubated at various pH levels was then measured at pH 7.2 by the manometric tech- nique. The results are given in Fig. 3.

One representative experiment may be described. An enzyme solution in 0.01 M phosphate buffer of pH 7.2 was used which hydrolyzed 10 gm. of acetylcholine per ml. per hour. Of this 0.2 ml. was diluted with 0.2 ml. of 0.1 M buffer of the desired pH and kept at 10” for about 30 minutes. 0.1 ml. of TEPP solution containing 0.2 y per ml. in the same buffer was

= 60 f

- t z

I 5 10 PH

FIG. 3. Inhibition of acetylcholine esterase by TEPP as a function of pH. The concentrated enzyme was exposed to TEPP at various pH values, then rapidly diluted, and the activity tested at pH 7. The symbols represent two different sets of data.

added and the mixture kept for 15 minutes. The mixture was rapidly diluted 500 times with gelatin-bicarbonate buffer. In the control the TEPP solution was replaced by buffer.

It is noteworthy that the act,ivity of the enzyme was not affected by exposure for a period of 1 hour t,o solutions with pH ranging from 5.5 to 10. The activity of the controls, when t8he pH was restored to 7.2, was equal in all cases. This indicates that the dependence of the enzyme activity on pH reflects reversible and not irreversible changes in the enzy- matic activity within the experimental period.

Fig. 3 relates the inhibitory effect of TEPP to pH. In view of the ex- tremely fast and irreversible reaction between enzyme and TEPP, the curve represents the true degree of reaction between the enzyme and the inhibitor.

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I. B. WILSON AND F. BERGMANN 485

TABLI~ III Inhibition of Acetylcholins E&erase by TEPP As Function of pH

The enzyme solution was diluted with buffer of the pH desired to give an activity of 53 mg. of acetylcholine split per ml. per ‘hour. 0.4 ml. of this solution was used for each test-tube. TEPP was prepared in a buffer solution containing 0.024 y per ml. Buffer was added to bring up the total volume to 4.1 ml. After 10 minutes in- cubation 3.6 mg. of acetylcholine in 0.9 ml. were added to each tube and readings taken 30 minutes later. 100 Klett units correspond to 1 PM of acetylcholine. The control without enzyme gave a reading of 450 Klett units. Addition of 1.2 ml. of TEPP did not change this value. Temperature 20’.

Estimated by graphic interpolation of these values, the amount of TEPP required for 50 per cent inhibition was 1.65 ml. for pH 7, 1.95 ml. for pH 8, and 2.4 ml. for pH 9. The CsO values were therefore 3.3,4.1, and 4.9 X 10-* M respectively.

TEPP

ml.

0 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4

T PH ‘I PH 8 PH 9

Klett units Per cent inhibition

246 0 208 15.4 186 24.4 172 30.1 160 36.0 152 38.2 129 47.6

Klett units

254 0

162 35.2 161 36.6 150 40.9 135 46.9 128 49.6 112 55.9 118 53.5

-

. _

-

Per cent inhibition

-

.-

-

Klett units

-

_ - Per cent

inhibition

187 0

137 26.7 122 34.8 108 42.3 120 35.8 105 43.9

-

30 t

f + ‘Q 0 +

- *

-:: I

20

0

IO

1J\ I I I I

5 6 7 8 9

PH

FIG. 4. Inhibitory action of TEPP as a function of pH. ~/CM represents the reciprocal of the molar concentration of TEPP required for 50 per cent inhibition.

In another group of experiments, graded amounts of TEFP in the ap- propriate buffer were added to an enz*vme solution diluted with the same

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486 CHOLINESTERASE. VII

buffer and the mixture was incubated for 10 minutes. Acetylcholine was then added and the enzymatic hydrolysis determined calorimetrically after 10, 30, and 60 minutes. In this set of experiments the activity of the enzyme was measured at the same pH as that at which the reaction with TEPP took place. Therefore, it represents a summation of the effect of pH on the interaction of the enzyme with TEPP and with acetylcholine. One typical experiment is described in Table III. Fig. 4 summarizes the data obtained in this way. The activity of TEPP is expressed as l/CbO; i.e., the reciprocal of the concentration required for 50 per cent inhibition. Each of these figures represents an average of several experiments. The difference in the percentage inhibition of acetylcholine hydrolysis between pH 7 and 9 is relatively small and the values at the alkaline side appear to be less accurate than at the acid side.

DISCUSSION

The data permit certain conclusions as to the nature of the active sur- face of acetylcholine esterase. They suggest the existence of an electrically negative site (group of atoms) and a part at which the ester linkage is broken and which therefore may be called the “esteratic” site.

The esteratic site contains a group of atoms which behaves as a base. The conjugate acid is not active. There is also a group of atoms which behaves as an acid, the conjugate base of which is inactive. These two functions may involve two separate groups within the site, or the dual rale may be pla,yed by the same group. This is represented by the fol- lowing scheme.

EH; +H+ +OH-

- EH - -- E- + Hz0

Inactive Active enzyme Inactive

These atomic groups may be part or the whole of the esteratic site. This view is supported by the following facts. (a) Acetylcholine and

TEPP do not undergo any changes with pH. Therefore, all changes of enzyme activity with pH must reflect changes in the protein structure. These changes permit the evaluation of the dissociation constants of the esteratic part, which will be reported in a subsequent paper. (b) The inhibitory action of TEPP shows an optimum between pH 7 and 8. Since TEPP is a neutral molecule, no attraction to the negative site is possible. The interaction with the prot,ein must therefore occur at the esteratic site and the occurrence of a pH optimum of TEPP inhibition thus reflects changes of the esteratic site. Moreover, the experiment with prostig- mine discussed below excludes any change in the negative site of the eR-

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P. B. WILSON AK‘D F. BERGMANN 487

zyme surface. The similar pH variation of acetylcholine hydrolykis sub- stantiates these conclusions.

The existence of an electrically negative site, at least for solutions which are not very highly acid (pH higher than 5.5), may be reasonably assumed a priori in view of the positive electrical charge of its normal substrate, acetylcholine cation. The following experimental data support this view. (a) The cationic form of the closely related substrate dimethylaminoethyl acetate is hydrolyzed at a much greater rate than the free base. (b) The cationic form of eserine is a much more potent inhibitor of acetylcholine esterase than the base.

The rBle of the positive charge in the cationic forms is mainly electro- static; that is, the positive charge and the changes in structure of the sub- strate which attend the conversion of base to acid are not involved in the forces which stabilize the critical complex except in so far as they increase t,he binding through electrostatic attraction. Stated in another manner, the reaction would be identical for both forms in a medium of infinite dielectric constant.

This view is supported by the following facts. (a) The dimethylamino- ethyl acetate base is hydrolyzed, although it cannot be strongly attracted to the anionic center. (b) The base form of eserine is an inhibitor, although again no strong electrostatic attraction is possible. This shows that eserine and prostigmine must interact not only with the negative but also with the esteratic site. The protecting effect of the two compounds against DFP reported previously (2) must be interpreted in the same way.

If it is accepted that the only change in passing from the free base of dimethylaminoethyl acetate to the cationic form is to add the electrostatic attraction, it is possible to apply the Brgnsted-Christiansen-Scatchard equation (6) and calculate the charge on the negative site and to estimate the distance of separation of the nitrogen and the negatively charged atom in the anionic center. From atomic radii this distance is estimated to be about 5 A.

The Br@nsted-Christiansen-Scatchard equation relates the constant for a complex between ions to the dielectric constant and the ionic strength of the medium as follows:

znzgc? znzez log K = log K’ - ~ -

2.3DkTr + 2.3DkT XI

1 + TK

where K is the affinity constant, K’ is the affinity constant for D = 00, K = 0, ZA and Z. are the number of electronic charges for ions A and B respectively, D is the dielectric constant, a is the electronic charge, k is the Boltzmann constant, T the absolute temperature, K = 1/(8re2/DkT)p, IL is the ionic strength, and r is the distance of closest approach.

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488 CHOLINESTERASE. VII

Let ZB apply to the enzyme and ZA = + 1 apply to dimethylaminoethyl acetate cation. F’or the free base Za = 0. Then

log K (base) ZB$ 1

K (cation) 5i-X

2.3rDkT 1 + ru

For cc = 0.2, water at 25”, this equation reduces to

2.1 x lo-‘0 T=

1% K (base)

K (cation)

where the value of D is to be estimated from the Schwarzenbach approx- imation (7). The data for eserine and dimethylaminoethyl acetate indicate a 20-fold greater affinity constant for the charged reactant compared to the free base. If 2, = -1, T = 6.5 X IO+ cm. The assumption of 2, = -2 would yield a larger value of T, namely 8.2 A, which is less satis- factory. Thus the negative site bears a single electronic charge. (The value for T is a little large, even for Zg = - 1. But it must be remembered that there is possibly a positive grouping in the neighborhood which would weaken the negative field. Also the data for pH 10 are subject to con- siderable experimental error, arising from the low values and from correc- tions for non-enzymatic hydrolysis. All considered, including the high ionic strength, the value derived for T is satisfactory.)

Whether the negative site of the active surface plays its r&e exclusively through electrostatic forces or, in addition, is involved in the formation of the critical complex leading to t.he breaking of the ester linkage cannot be decided at present and requires further investigation.

The group of phosphates, which are active inhibitors, like TEPP, DFP, and parathion, differs from that of the similarly constituted but inactive phosphates in that during hydrolysis a bond involving a phosphorus atom is ruptured in an SN2 mechanism. The enzyme attacks the phosphates in a nucleophilic substitution reaction, and therefore the part of the inhibitor entering the covalent bond with the enzyme has in all cases the structure [(RO> zPOl+. In the other phosphates a C-O bond is always broken during hydrolysis.

The proposed mechanism explains the decrease of inhibitory activity at low pH at which the esteratic site is occupied more and more by H ions. However, the explanation for the decrease of inhibitory action at the alkaline side must await further investigation of the esteratic group.

SUMMARY

The inhibition of acetylcholine esterase by various inhibitors was inves- tigated as a function of pH and related to the electric charge of the inhibi-

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I. B. WILSON AND F. BERGMANN 489

tor. These observations have revealed several features of the active sur- face of the enzyme.

1. Prostigmine cation inhibition is not affected by pH in the range tested (6 to 10).

2. Eserine inhibits strongly at pH 6. The inhibition decreases with in- creasing pH. Eserine cation is a stronger inhibitor than the base. How- ever, at pH 10 the entire inhibition must be ascribed to the base, since the proportion of cationic form present at this pH was found to be negli- gible.

3. The rate of hydrolysis of dimethylaminoethyl acetate by acetylcholine esterase was tested at pH 6 to 10 and compared with the pH dependence of acetylcholine hydrolysis. The compound was found to exist in cationic and basic forms, depending upon the pH of the medium. The cation was found to be the more active form, but the neutral molecule was also hydro- lyzed, though at a much lower rate.

4. TEPP inhibition shows a definite optimum between pH 7 and 8. This fact was demonstrated in two ways: (a) by measuring the inhibition at various pH values and (b) by exposing concentrated enzyme to the inhibitor at various pH values and determining at neutral pH the activity remaining after high dilution.

The data reveal that the active surface contains a group of atoms bear- ing a single negative charge. The active surface also contains an esteratic site which behaves in acid solution as a proton acceptor, in alkaline solution as a proton donor. Both the conjugate base and acid are inactive. The esteratic site is subject to electrophilic attack. This may explain the mechanism by which TEPP and similar phosphates, able to transfer phos- phonium ions, combine with the enzyme.

The authors wish to express their gratitude to Dr. D. Nachmansohn for his inspiring guidance and advice throughout this work. They also wish to thank Mrs. Ida Freiberger for her assistance in performing the experi- ments.

BIBLIOGRAPHY

1. Nachmansohn, D., Rothenberg, M. A., and Feld, E. A., J. Biol. Chem., 174,247 (1948).

2. Augustinsson, K.-B., and Nachmansohn, D., J. Biol. Chem., 179, 543 (1949). 3. Rothenberg, M. A., and Nachmansohn, D., J. Biol. Chem., 166, 223 (1947). 4. Hestrin, S., J. Biol. Chem., 160, 249 (1949). 5. Jones, L. W., and Major, R. T., J. Am. Chem. Sot., 62,307 (1930). 6. Hestrin, S., Biochim. et biophys. acta, 4, 310 (1950). 7. Schwarzenbach, G., 2. physik. Chem., Abt. A, 176, 133 .(1936).

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Irwin B. Wilson and Felix BergmannINHIBITORS

DERIVED FROM EFFECTS OF pH ON ACETYLCHOLINE ESTERASE

THE ACTIVE SURFACE OF STUDIES ON CHOLINESTERASE: VII.

1950, 185:479-489.J. Biol. Chem. 

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