STUDIES ON CHOLINESTERASE

VI. KINETICS OF THE INHIBITION OF ACETYLCHOLINE ESTERASE*

BY KLAS-BERTIL AUGUSTINSSONt AND DAVID NACHMANSOHN

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

(Received for publication, December 20, 1948)

Enzyme inhibitors have long been used in the analysis of cellular func- tion for separating different steps in complex chemical reactions and for studying the effect of blocking these reactions in the intact cell. Inhibi- tors of choline ester-splitting enzymes, especially the alkaloids prostig- mine and eserine, have attracted the interest of investigators in view of the physiological rale of acetylcholine. In contrast to the inhibitors pre- viously known, some powerful agents recently developed inactivate the enzymes irreversibly. This property has made it possible to approach many new problems for which the compounds previously available were not appropriate. Particularly, the diisopropyl fluorophosphate (DFP), in the presence of which the irreversible enzyme inactivation is a relatively slow process, has been an excellent tool for testing the essentiality of acetylcholine-hydrolyzing enzymes in nerve conduction (l-3).

In addition, the new compounds offer an opportunity for studying whether the signs of toxicity must be attributed exclusively to interaction with the enzymes. As such an interaction depends on many factors, in vitro and still more in viva, it is imperative to study the kinetics of the inhibition of specific esterases, the inactivation of which appears to be primarily responsible for the toxic symptoms (4, 5). The properties of the esterases of conductive tissue and erythrocytes are distinctly different from other choline ester-splitting enzymes (6, 7). The term acetyl- choline esterase (ACh-esterase) has been proposed (8) for this type of esterases.

In recent studies on the kinetics of the inhibition of ACh-esterase some differences between DFP and the alkaloids have been described (9, 10). As before, the ACh-esterase used was exclusively the highly purified preparation obtained from the electric tissue of Electrophorus electricus.

* The work has been carried out under a grant from the United States Public Health Service.

t Fellow of the American-Scandinavian Foundation, with additional grants from the Swedish National Medical Research Council and the United States Public Health Service. Present address, Biochemical Institute, University of Stockholm.

543

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544 CEIOLINESTERASE. VI

Methoda

The enzyme preparation, obtained as previously described (ll), had an activity equivalent to 20 gm. of acetylcholine chloride split per hour per ml. of solution. The protein content was 1 mg. per ml. The inhibitors used, prostigmine bromide, eserine sulfate, DFP, and tetraethyl pyro- phosphate (TEPP), were freshly prepared for each experiment. They were dissolved in the same buffer solution, containing gelatin, as the en- zyme. In the experiments in which the inhibitor effects were tested with various substrate concentrations, the final molar concentrations of acetyl- choline were 1.1 X 10-r, 3.3 X lO-*, 1.1 X lO-*, 3.3 X HY, 1.1 X 10m3, 3.3 X lo-‘. In most of the other experiments, the acetylcholine concen- tration was 3.3 X 10-’ M, which is close to the optimum. The incuba- tion of the enzyme with TEPP at lo’, the subsequent dilution, and the manometric determinations of the enzyme activity with the Warburg method were carried out as described in a preceding study (10). The same procedure was also used in the experiments in which the protective action of prostigmine and eserine against DFP and TEPP was tested. The course of the enzymatic hydrolysis of acetylcholine in the presence of inhibitors and in various substrate concentrations was determined mano- metrically at 23-24’ in the modification described (7).

In Paper V (10) only the enzyme inhibition by DFP was analyzed and compared with the kinetics of the inhibition by the alkaloids prostigmine and eserine. The present experiments include tetraethyl pyrophosphate, found by DuBois and Mangun (12) to be a very potent inhibitor of choline ester-splitting enzymes.

Irreversibility of TEPP Eflect-The action of this compound on ACh- e&erase is almost immediately irreversible (Table I). This has been tested with the dilution method described for the DFP action. The con- centrated enzyme solution was incubated with TEPP at 10” for varying periods of time, diluted 5000 times, and tested for enzymatic activity. This dilution brought the concentration of TEPP into a completely in- effective range. When the enzyme is incubated with DFP under similar conditions, the activity may at first be completely restored, but gradu- ally the reactivation becomes less complete. At 10” it may take 2 to 3 hours until the process becomes completely irreversible. In contrast, the TEPP effect appears to be complete almost immediately. After only 2 minutes incubation at low temperature, no reactivation was obtained by dilution, and after 120 minutes incubation the effect for the various con- centrations used was about the same as after 2 minutes. The concentra-

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K.-R. AUGIJSTINSSON AND D. NACHMANSQHN 545

tion of TEPP required for a 50 per cent inhibition is much lower than in the case of DFP, the inhibitory effect thus being apparently not only faster but also stronger.

Inhibition by TEPP As Function of Enzyme Concentration-Although the action of TEPP is much stronger than that of DFP, a considerable excess of inhibitor over enzyme concentration is necessary for producing 50 per cent inhibition. In the most concentrated solution, the enzyme concentration during incubation (Table II) is about 4 X NV’ M, esti-

TABLE I Irreversible Inactivation oj ACh-esterase by TEPP

The enzyme wza incubated with TEPP for varying periods of time at 10”. Sub- sequently, the solution was diluted and the enzyme activity determined. ba, = enzymatic hydrolysis expressed in J. of CO2 evolved per 30 minutes.

TEPP coxlcentration

Y x 10

Control 0.69 1.88 1.28 1.38 2.70

Control 0.69 0.69 1.38 1.38 1.38 1.38 1.38 2.70 2.76

- I

60 16 30 60 60

60 120

2 4 8

60 120 60

120

baa Per cent inhibition

195 148 98

116 92 67

24 50 41 53 66

199 146 165 108 107 107 106 100 39 14

27 22 46 46 46 47 50 80 93

mated on the assumption of a molecular weight of about 3 million. This figure is calculated on the basis of the sedimentation rate in the analytical run in which only one component was present (11). In this concentra- tion, TEPP in 1.4 X 10-s M concentration produces 50 per cent inhibi- tion after 2 minutes incubation. The inhibitor is thus about 35 times in excess of the enzyme. With increasing dilution of the enzyme, the excess of inhibitor required for obtaining 50 per cent inhibition increases. In the greatest dilution tested, the excess is close to 3000 times. In the case of DFP, the excess in the highest concentration used was about the same. as with TEPP. In the greatest dilution, however, the excess was

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546 CHOLINESTERASE. VI

more than 100,000 times. Much longer incubation periods were used in the case of DFP, and the results are therefore not comparable.

Course of Inhibitor Reactions-The course of the enzymatic hydrolysis of acetylcholine in the presence of the two types of inhibitors (reversible and irreversible) has been studied for various inhibitor concentrations

TABLE II Inhibition of ACh-esterase by TEPP As Function of Varying Enzyme Concentrations

The enzyme in varying dilutions was incubated for a period of 5 minutes at 10” with different inhibitor concentrations. Subsequently, the enzyme was diluted to a concentration 1:5000 of the original solution and the activity determined. The experiments with the lowest dilution, 1:5000, were incubated directly in the Warburg vessel. ba as in Table I.

Enzyme concentration Inhibitor concentration

Undiluted

10 X dilution

100 x “

1000x “

5ooox “

-7 T Experiment 1

Y

Control 6.9 X 10-T

13.8 X lo-’ 27.6 X 10-T Control

1.7 x lo-’ 3.45 x lo-’ 6.9 x 10-T

Control 2.15 X lo-’ 4.3 x 10-a 8.6 x 10-S

17.2 X lo+ Control 4.3 x lo-’ 8.6 x 10-8

Control 0.26 x 10-e 0.7 x lo-’ 2.3 x 10-0 4.6 X lo-’

ho

195 148 92 67

186 102 44 0

186

92 47 17

297 75 23

200 188 154 100

Per cent inhibition

24 53 66

46 76

100

51 75 91

68 89

6 23 50

-

-

Experiment 2

a:,

199 146 106 39

184 84 40

183 133 102 55 18

207

103 63

Per cent inhibition

27 47 82

54 78

22 44 70 90

50 70

(Figs. 1 to 4). In one series of experiments, the enzyme was incubated with the inhibitor prior to the addition of acetylcholine (A); in a second series, inhibitor and substrate came into contact with the enzyme simul- taneously (B). The optimum acetylcholine concentration was used (3.3 x 10-J M).

Prostigmine-When the ensyme is incubated with various concentra-

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K.-B. AIJOUSTINSSON AND D. NACHMANSOEN 547

tions of prostigmine for 70 minutes before the addition of acetylcholine, it is 10 to 25 minutes until equilibrium is reached, the time depending upon the inhibitor concentration used (Fig. 1, A). During this period, the inhibition is stronger. Without incubation, the period of time re- quired for reaching the equilibrium is about the same, but during this period the inhibition is less strong than in equilibrium. In one case, the inhibitor reacts with the enzyme before the substrate is present; in the other case substrate and inhibitor compete for the enzyme. It is there-

Y ( ) 20 40 60

TIME (min.)-

Wa. 1. The course of hydrolysis of acetylcholine in the presence of various con- centrations of prostigmine. A, enzyme incubated 70 minutes with the inhibitor before the addition of acetylcholine (final concentration 3.3 X 10-z M). B, inhibitor and acetylcholine simultaneously mixed with the enzyme. The dotted line refers to non-enzymatic hydrolysis. Curve 1 is the control, Curves 2 to 6, the hydrolysis in the presence of prostigmine in 6.25, 12.5, 25, 50, and 100 X 10-’ M concentration (during incubation 5 times higher).

fore not surprising that the initial reaction velocities in the two types of experiments are different. However, after equilibrium the reaction ve- locities are the same for the same concentration of prostigmine. This is consistent with previous observations that prostigmine inactivates ACh-e&erase completely reversibly.

The decrease of the reaction velocity at the end of the control experi- ment is due to the low concentration to which the acetylcholine has been reduced by the hydrolysis. The total amount of COlr which can be evolved from the acetylcholine solution used is 148 ~1.

Eserine behaves similarly to prostigmine. The period of time until

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548 CHOLINESTERASE. VI

the equilibrium is reached depends upon the concentration of the in- hibitor. In equilibrium, the inhibition is the same in the experiments with and without incubation (Fig. 2). As shown by other authors, the reaction between eserine and ACh-esterase is completely reversible.

In cases in which the reactions were followed for a longer time (2 hours and more), the inhibition was slower. This may be due to inactivation of eserine, observed by Ellis and coworkers (13) in experiments with serum cholinesterase.

aJ

0 20 40 60 0 20 40 60

TIME(min.)+

FIG. 2. The course of hydrolysis of acetylcholine in the presence of various con- centrations of eserine. A, enzyme incubated 66 minutes with the inhibitor before the addition of acctylcholine (final concentration 3.3 X lo+ M). B, inhibitor and acetylcholine simultaneously mixed with the enzyme. Curve 1 is the control, Curves 2 to 6, the hydrolysis in the presence of eserine in 6,12,24,48, and 96 X 10-7~ concentration (during incubation 5 times higher).

DFP-As pointed out, the fundamental difference between the inhibi- tion of ACh-esterase by DFP and that by prostigmine and eserine is the reversible nature of the latter inhibition. When the enzyme was incu- bated with DFP for 60 minutes at 1.25 X lo+ M, it lost 50 per cent of its activity (Fig. 3). When the enzyme was mixed simultaneously with acetylcholine and DFP, without incubation, much higher inhibitor con- centrations were necessary for obtaining 50 per cent inhibition. At a concentration of lO+ M DFP, no inhibition at all was observed during the first 40 minutes. After that period, the activity decreased slowly. With lo-’ M DFP, the inhibition started earlier but no real equilibrium was

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K.-B. AUGUSTINSSON AND D. NACHMANSOHN 549

reached. Only when the DFP concentration was as high as lo-* M was the inhibition nearly complete.

The results suggest that the acetylcholine protects the enzyme against DFP. However, the acetylcholine concentration (3.3 X 10e3 M) is 30 times as high as the inhibitor concentration in the first experiment (1O-6 M DFP), and 3 times as high in the second experiment. On the other hand, in the experiment in which the inhibition of the hydrolysis was

TIME( min.)+

IO

FIG. 3. The course of hydrolysis of acetylcholine in the presence of various con- centrations of DFP. A and B, as in Fig. 2, but acetylcholine concentration in B = 1 X 10-S M. Curves 1 and 7 are the controls, Curves 2 to 6, the hydrolysis in the presence of DFP in 1.25,2.6,&O, 8.75, and 10 X 10-T M concentration, Curves 8 to 10 in 1.5, 10, and 100 X 10-S M concentration.

nearly complete, the concentration of DFP was 3 times as high as that of acetylcholine.

A much stronger protective action may be obtained with prostigmine. This compound may protect the enzyme against DFP if present in the same concentration (Table III!). The enzyme was incubated with pro- stigmine for 20 minutes at 10’ prior to the addition of DFP. The ac- tivity of the enzyme after 150 minutes incubation with DFP may be restored partly or nearly completely by dilution (5000 times). Without prior incubation with prostigmine, 50 per cent of the enzyme was irre-

1 These experiments were carried out in collaboration with Dr. M. A. Rothenberg.

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550 CHOLINESTERASE. VI

versibly inactivated. With prostigmine at half the concentration of DFP, the protection was half as strong. At room temperature the pro- tective effect of prostigmine seems less complete. Incubation with

TABLE III Protective Effect of Prostigmine against Action of DFP on ACh-esterase

The enzyme solution was incubated for 150 minutes with DFP (final concentra- tion 1.6 X 10-a M), then diluted about 5000 times, and the activity was measured. Prostigmine (or eserine) was added to the solution 20 minutes prior to DFP. The compounds were added always in 0.1 ml. to 0.1 ml. of enzyme solution; the total volume was in all cases 0.3 ml. The final concentration of prostigmine (or eserine) was the same as that of DFP (1.6 X 10-e M) except in one case, 3.2 X lo-‘ M (2 X) and in two cases 0.8 X 10-O M (4 X). ba = enzymatic hydrolysis expressed in ~1. of CO* per 30 minutes. Experiments 1 to 3, at 10’; Experiment 4, at 23”. -

Compound bie

Control ..................................... DFP.. ..................................... Prostigmine + DFP ........................

“ ................................. Eserine + DFP .............................

I‘ .....................................

131 65

131 145 85

137

Control .................................... DFP ...................................... Prostigmine + DFP ....................... (4 X) Prostigmine + DFP ................. Prostigmine ...............................

115 60 92 78

10s

Control .................................... 238 DFP ...................................... 118 (2 X) Prostigmine + DFP ................. 218 Prostigmine + DFP ....................... 224 (3 X) Prostigmine + DFP ................. 178

Control. ................................... DFP ...................................... Prostigmine + DFP .......................

“ ............................... Eserine + DFP ............................

“ ....................................

210 55

131 265 77

203 _-____--- -

Per cent inhibition

50.5 0 0

35.0 0

48.0 20.0 32.0

6.0

50.5 8.5 6.0

25.0

74.0 37.5

2.5 63.5

3.5

eserine protects the enzyme much less against subsequently added DFP than does incubat,ion with prostigmine. This finding is in agreement with the observations of Koelle (14) that eserine has a protecting effect against the action of DFP in brain homogenates.

TEPP inhibits the enzyme activity in a manner similar to DFP, but

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K.-B. AWIUSTINSSON AND D. NACHMAh%OHN 551

much more strongly. On incubation for 60 minutes with 8 X lo-+ M

TEPP, the inhibition is 50 per cent (Fig. 4, A). In experiments with- out incubation, the acetylcholine seems to protect the enzyme against the action of TEPP and no equilibrium is reached between inhibitor, substrate, and enzyme. A weak effect is obtained with a concentration of 3.5 X lo--’ M, which is 125 times higher than that producing 50 per cent inhibi- tion after incubation. The inhibitory effect progresses with time.

0 20 40 60 80

TIME(min)w

FIG. 4. The course of hydrolysis of acetylcholine in the presence of various con- centrations of TEPP. A and B, aa in Fig. 2. Curve 1 is the control, Curves 2 to 11, the hydrolysis in the presence of TEPP in 0.7, 1.4, 2.8, 5.6, 11.2, 87.6, 175, 350, 700, and 14CO X lo-’ M concentration.

The enzyme was also incubated with prostigmine or eserine prior to the addition of TEPP and the activity then tested to subsequent dilution. No protective action was observed if the two types of inhibitors were added in the same concentration.

Inhibition As Function of Inhibitor Concentration-The inhibition as a function of inhibitor concentration may be analyzed by plotting u/u’ against the concentration of the inhibitor, v being the reaction velocity in the absence of the inhibitor I, v’ in its presence. In the case of competi- tive inhibition and constant concentrations of enzyme and substrate, a straight line is thereby obtained, according to Equation 1.

(1)

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552 CHOLINESTERASE. VI

[S] and [I] are the concentrations of substrate and inhibitor respectively; Kg and Kr represent the dissociation constants of the complexes between enzyme and substrate and enzyme and inhibitor respectively. The inter- cept of the straight line on the ordinate (v/v’) is 1. From the slope of the line (i.e., Ks/Kr([S] + KS) and known values of [S] and K8, KI can be calculated. This method has been applied by Augustinsson (15) with satisfactory results for the analysis of the action of choline on various esterase systems and has therefore been used in the present studies.

12 I I I I I I I I .

IO -

4-

t 2- dX

‘> I /

3 . 1 ’ I I I I I I I _ I 2 4 6 8 IO 12 14 I6

Rel.111 --+ FIQ. 5. Inhibition of ACh-e&erase by prostigmine ss function of inhibitor con-

centration. u = velocity in absence, u’ in the presence of inhibitor, expressed in ~1. of COII evolved in 30 minutes @SO). Acetylcholine concentration, 3.3 X lOma M;

inhibitor concentration, relative inhibitor concentration (Rel.[A), 1 = 6.25 X 10-7M.

l , without incubation; 0, with incubation; X, values obtained for the above concentration in the experiments with varying substrate concentrations (Fig. 9). KI = 1.6 X 10-7.

Prostigmine and Eserine-Applying this method of analysis for the ac- tion of prostigmine and eserine on ACh-esterase results in straight lines (Figs. 5 and 6). The data are the same in experiments with and without incubation. This indicates that the two alkaloids inhibit the esterase activity competitively; i.e., that they react reversibly with the same active center of the enzyme as does acetylcholine.

The affinity of the enzyme is 2.6 times higher for eserine than for pro- stigmine. The dissociation constants for the enzyme inhibitor complexes are 1.6 X lo-’ and 6.1 X lo-* respectively, calculated according to Equation 1.

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K.-B. AUGUSTINSSON AND D. NACHMANSOHN 553

FIQ tion. 6.0 x

FIQ. 6. Inhibition of ACh-esterase by eserine as function of inhibitor concentra- . 6. Inhibition of ACh-esterase by eserine as function of inhibitor concentra- tion. Description as in Fig. 5. Relative inhibitor concentration (Rel. [A), 1 = Description as in Fig. 5. Relative inhibitor concentration (Rel. [A), 1 = 6.0 x 10-T M. Kr = 6.1 X lo-*. 10-T M. Kr = 6.1 X lo-*.

28 28 I I I I i i I I I I I I I I I I

24 - 24 -

20 - 20 -

18 _ 18 _

12 - 12 -

I I I I I I I I I I I I I I 012 012

Rol.CIl ,* ,* 6 6 8 10 12 14 16 8 10 12 14 16

Rol.CIl

8-

30 11 I I I I I I I 0 1 2 4 6 8 IO 12 14 16

Rel CII - FIG. 7. Inhibition of ACh-esterase by DFP and TEPP respectively as a function

of inhibitor concentration. Description as in Fig. 5. Relative inhibitor concentra- tion (Rel. [I]), 1 = 2.5 X 10-T M DFP and 2.8 X lO+ M TEPP respectively.

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554 CHOLINESTERASE. VI

DFP and TEPP-Both DFP and TEPP behave differently from the alkaloids. By plotting the degree of inhibition expressed by V/V’ against the inhibitor concentration, straight lines are not obtained (Fig. 7). With increasing DFP and TEPP concentrations, the inhibition increases more rapidly than is expected in a competitive inhibition. The curves in Fig. 7 are based on the data obtained when the enzyme had been in- cubated for 1 hour with the inhibitors.

IO 9 8 7 6 5

FIG. 8. Per cent inhibition of ACh-esterase by prostigmine, eserine, DFP, and TEPP, respectively, as function of the negative log of the molar concentration of the inhibitors (~1). The dotted line refers to the effects obtained with prostigmine and eserine when measured immediately after addition of acetylcholine before equilib- rium is reached.

A picture of the relative strength of the four inhibitors studied is ob- tained if the per cent inhibition is plotted against ~1, the negative log of the molar concentration of inhibitor (Fig. 8). The dotted line indicates the values obtained if the activity of the two alkaloids is measured during a period of 20 to 30 minutes after addition of acetylcholine following incubation. As pointed out before, during that period the equilibrium has not yet been reached and these values, used in the preceding study (lo), indicate a stronger inhibition than that obtained in equilibrium.

Inhibition As Function of Substrate Concentration-The inhibition of ACh-esterase at various substrate concentrations by the four inhibitors is shown in Figs. 9 to 12. The enzyme was incubated for 60 minutes with the inhibitor before determining the activity.

In presence of prostigmine, the optimum substrate concentration is changed to a higher concentration in the presence of the drug (Fig. 9).

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K.-B. AUGUSTINSSON AND D. NACHMANSO~ 555

With increasing inhibitor concentration, this shift becomes increasingly stronger. The degree of inhibition for a given inhibitor concentration therefore varies greatly with the substrate concentration. At 3.3 X lo-* M acetylcholine, the enzyme is inhibited 10 per cent by 2 X lo4 M prostigmine; at 3.3 X 10m3 M acetylcholine, the inhibition by the same concentration of prostigmine is as high as 88 per cent.

,

5

2

4 3 2 I c- PS

FIG. 9

100

75

50

25

t 24

P 0

/

,

I_ 4 3 2

4---J PS FIG. 10

FIG. 9. Activity-p8 curves for the enzymatic hydrolysis of acetylcholine by ACh- esterase in the presence of various concentrations of prostigmine. Curve 1 is the control, Curves 2 to 5, the hydrolysis in the presence of prostigmine in 0.4.1,2, and 10 X IO+ M concentration.

FIG. 10. Activity-pS curves for the enzymatic hydrolysis of acetylcholine by ACh-esterase in the presence of various concentrations of eserine. Curve 1 is the control, Curves 2 to 4, the hydrolysis in the presence of eserine in 3,6, and 18 X WY M concentration.

Eserine-According to the results demonstrated in Fig. 6, eserine, like prostigmine, inhibits acetylcholine competitively. However, the opti- mum substrate concentration is only slightly changed in the presence of low eserine concentrations. The shift becomes more pronounced at rela- tively high eserine concentrations. In that case, the optimum substrate concentration is definitely higher than in the absence of the inhibitor (Fig. 10). This agrees with the finding that in the presence of 3.6 X lo4 M eserine the optimum acetylcholine concentration (pS) for the erythrocyte esterase of horse blood is 1.5, whereas it is 2.6 in the absence of the inhibi- tor (15).

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556 CHOLINESTEFUSE. VI

DFP and TEPP-In the case of the irreversible inhibitors, the inhibi- tion is independent of the substrate concentration. This is illustrated in Figs. 11 and 12. At a concentration of 5 X lo-’ M DFP, for example, the enzyme is inhibited to about 70 per cent at all substrate concen- trations.

100 I I I

75

50

25

T

8 Alo

4 3 2 1 - PS

FIG. 11

100

75

25

T

I% fl0

I I I

3 2 r’ps

FIG. 12

FIG. 11. Activity-pS curves for the enzymatic hydrolysis of acetylcholine by ACh-esterase in the presence of DFP. Enzyme incubated 60 minutes with the in- hibitor before addition of acetylcholine. Curve 1 is the control, Curves 2 and 3, the hydrolysis in the presence of 1 and 2 X 10-’ M concentration of DFP (during incubation; during determination, the concentration was 5 times lower).

FIG. 12. Activity-pS curves for the enzymatic hydrolysis of acetylcholine by ACh-e&erase in the presence of TEPP. Enzyme incubated 60 minutes with the inhibitor before addition of acetylcholine. Curve 1 is the control, Curves 2 and 3, the hydrolysis in the presence of 5.6 and 8.4 X 10-O M concentration of TEPP (during incubation; during determination, the concentration was 5 times lower).

DISCUSSION

Several new differences between the various inhibitors of choline ester- splitting enzymes have been revealed in addition to those described pre- viously (10). The sequence in which inhibitor and substrate come in contact with the enzyme affects the course of hydrolysis in the case of the reversible inhibition, the alkaloids prostigmine and eserine, in a way different from that found in the case of DFP and TEPP. Whether the enzyme is first incubated with alkaloids or whether substrate and inhibi-

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K.-R. AUGUSTINSSON AND D. NACHMANSOHN 557

tor are added simultaneously, the degree of inhibition is the same after equilibrium has been reached. Before the attainment of an equilibrium, a period which lasts 10 to 25 minutes, depending upon the concentration used, the inhibition is stronger than during equilibrium when the enzyme has been incubated with the inhibitor. Since both compounds are com- petitive inhibitors of the enzyme and the formation of the enzyme com- plex in both cases is completely reversible, it should be expected that the equilibrium is the same irrespective of incubation. The substrate con- centration is much higher than the inhibitor concentration and this may explain why, in the case of simultaneous addition, the inhibition is less strong in the beginning than later, whereas if the enzyme has formed a complex with the inhibitor before the substrate is added, the inhibition is stronger in the beginning. When v/v’, the ratio of the velocity of the hydrolysis in absence and presence of the inhibitor, is plotted against in- hibitor concentration, the same straight line is obtained with or without incubation with both alkaloids. But the inhibitory effect of eserine is about 2.6 times stronger than that of prostigmine.

In the case of irreversible inhibition, the degree of inhibition depends upon the time of incubation, as was previously demonstrated for DFP (10). Therefore, no equilibrium is attained, and if v/v’ is plotted against inhibitor concentration, the line is not straight. The same is true for TEPP, which, like DFP, inactivates the enzyme irreversibly, though more rapidly. If acetylcholine is added before the inhibitor, much higher concentrations of the latter become necessary for obtaining the same degree of inhibition as that observed after incubation. This protective effect of acetylcholine against the action of these two compounds contain- ing organic phosphorus suggests that these compounds act on the same active center of the enzyme molecule as acetylcholine. This assumption is supported by the observation that prostigmine in the same concentra- tion as the DFP has a very strong protective action. The protective action is obtained with very low concentrations if compared with that of acetylcholine. This may be explained by the high affinity of prostigmine to the enzyme. The affinity of DFP to the enzyme cannot be evaluated since there is no equilibrium, but, as far as one can speak of affinity, it appears to be of a similar order of magnitude as that of prostigmine. Eserine in spite of its stronger inhibitory effect protects the enzyme markedly less against DFP. As long as the exact reaction is unknown, a satisfactory explanation for this difference will be difficult. The lack of any protective action by prostigmine against TEPP if equimolecular concentrations are used is not surprising. It may be explained by a much higher afllnity of TEPP to the enzyme, since it inhibits in so much lower concentrations. It would be interesting to test whether a protec-

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558 CHOLINESTERASE. VI

tive action of prostigmine may be obtained in 100 to 1000 times higher concentrations, but the concentration of the available enzyme prepara- tion is too low for such an experiment.

Although it is possible to conclude, on the basis of the data presented, that both types of inhibitors may act on the same center of the enzyme as acetylcholine, but that in one case they form a reversible and in the other an irreversible complex, the underlying chemical reactions are at present unknown. This situation may be analogous to the inhibition of the combination of hemoglobin with oxygen by carbon monoxide and by potassium ferricyanide. Both inhibitors combine with the iron of the prosthetic group, the iron porphyrin. The carbon monoxide effect is easily reversible, whereas the potassium ferricyanide transforms the iron irreversibly.

Study of the competitive inhibition of the enzyme by prostigmine and eserine has revealed another difference between the two alkaloids. Pro- stigmine causes a marked increase of optimum substrate concentration with increasing inhibitor concentration. It has been demonstrated for erythrocyte and brain esterase that choline exerts a shift of the opti- mum substrate concentration; the more choline is added, the higher is the optimum substrate concentration and the lower the activity. It is possible that other quaternary ammonium ions may behave similarly and that such compounds may inhibit competitively the formation of the enzyme-substrate complex.

In the case of eserine, the shift of optimum concentration is much less pronounced and is marked only in high concentration of the inhibitor. Therefore, in low (e.g., W4 M) acetylcholine concentration, both alkaloids inhibit the enzyme at the same concentration to about the same degree; in high (10-l M) acetylcholine concentrations, on the other hand, prostig- mine does not affect the enzyme activity at all, whereas eserine has a strong effect. The absence of a shift of optimum in the case of DFP and TEPP may be explained by a gradually lowered activity, since these compounds inhibit the enzyme irreversibly.

SUMMARY

The study of the kinetics of the inhibition of ACh-esterase by two types of inhibitors, reversible and irreversible, has been continued.

TEPP was found to inactivate the enzyme irreversibly, and at 10” almost immediately, in contrast to DFP with which, at this temperature, the irreversible reaction is a relatively slow process. The study of the action of this inhibitor as a function of enzyme concentration has revealed that a considerable excess of inhibitor is necessary, which increases with increasing dilution. The enzymatic hydrolysis of acetylcholine in the

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K.-B. AUGUSTINSSON AND D. NACHMANSOHN 559

presence of prostigmine and eserine attains an equilibrium which is the same whether or not the enzyme has been incubated with the inhibitor prior to the addition of acetylcholine. No such equilibrium is attained with the irreversible inhibitors, DFP and TEPP. If acetylcholine is added to the enzyme simultaneously with the inhibitor, a protective action is observed, suggesting that all four inhibitors act on the same active center of the enzyme. TEPP is by far the strongest of all four inhibitors tested.

When the inhibitor effects are studied at various substrate concentra- tions, prostigmine and, to a lesser degree, eserine produce a shift of the optimum substrate concentration. No such shift is observed in the case of the two irreversible inhibitors.

We want to express our thanks to Mrs. Emily Feld-Hedal and to Mrs. M. Augustinsson for their assistance in the experiments.

BIBLIOGRAPHY

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NachmansohnKlas-Bertil Augustinsson and DavidACETYLCHOLINE ESTERASE

KINETICS OF THE INHIBITION OF STUDIES ON CHOLINESTERASE: VI.

1949, 179:543-559.J. Biol. Chem. 

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