Procaine as a substrate and possible allosteric effector of cholinesterases

Neurochemistry International, Vol. 5. No. 5, pp. 559 569, 1983 Printed in Great Britain. All rights reserved

0197 0186/83 $ 3 . 0 0 + 0 . 0 0 ~ 1983 Pergamon Press Ltd

P R O C A I N E AS A SUBSTRATE A N D POSSIBLE ALLOSTERIC E F F E C T O R O F C H O L I N E S T E R A S E S R. M. DAWSON and M. PORETSKI Materials Research Laboratories, Department of Defence Support, P.O. Box 50, Ascot Vale, Victoria 3032, Australia (Received 8 November 1982; accepted 12 January 1983)

Abstract The local anaesthetic procaine showed the properties of an allosteric effector of bovine erythrocyte acetylcholinesterase at low ionic strength: it antagonised inhibition of substrate hydrolysis caused by decamethonium, decreased the rate of ageing of isopropylmethylphosphonyl-acetylcholinesterase, increased the rate of decarbamylation of dimethylcarbamyl-acetylcholinesterase, and interacted synergistically with the nucleophilic alcohol 3,3-dimethyl-l-butanol in the acceleration of decarbamylation. These allosteric effects almost completely disappeared as the ionic strength was increased to a physiological level, and they could not be demonstrated at the physiological ionic strength with membrane-bound human erythrocyte acetylcholinesterase. There was no evidence of significant cooperativity in the binding of procaine to the enzyme, nor in the binding of the substrate acetylthiocholine in the presence of procaine, contrary to reports in the literature for other sources of acetylcholinesterase. Procaine was not hydrolysed by acetylcholinesterase (EC 3.1.1.7) although it is a substrate for serum cholinesterase (EC 3.1.1.8). The possibility that the results at low ionic strength can be explained on the basis of procaine binding to the active site of acetylcholinesterase (at low concentrations) and also to a peripheral allosteric site (at higher concentrationst is discussed. The results confirm the complexity of the kinetics of acetylcholinesterase, and extend the range of compounds with the ability to modify rates of decarbamylation and ageing.

In media of low ionic strength, additional binding sites for various cationic ligands, physically distinct from the active catalytic centre, have been demonstrated by kinetic measurements and by measurements of direct binding of fluorescent ligands (Rosenberry, 1975; Berman et al., 1981). It has been proposed that the conformation of the enzyme changes in response to ligand binding at these peripheral sites (allosteric sites) with a consequent increase in catalytic activity, e.g. increased rates of carbamylation, decarbamylation, deacetylation and oxime-induced dephosphorylation (Rosenberry, 1975; Berman et al., 1981; Bolger and Taylor, 1979; Tomlinson et al., 1980). Decreased rates of ageing of phosphonylated AChE are probably another manifestation of the conformational change (Crone, 1974). Most peripheralsite effects on the catalytic activity are abolished or require much higher concentrations of effector at a physiological ionic strength (Rosenberry, 1975; Changeux, 1966). The interaction of the local anaesthetic procaine with the cholinergic nervous system has been studied by a number of workers (e.g. Wills, 1963; Foidart and Gridelet, 1974; Roufogalis and Wickson, 1975; Israel and Meunier, 1979; Tsai et al., 1979) but no allosteric

The active catalytic centre of acetylcholinesterase (ACHE, acetylcholine acetylhydrolase; EC 3.1.1.7) consists of two subsites; a trimethylammonium binding site, which is involved in the binding of substrates and inhibitors, and an esteratic site. The latter site includes a serine hydroxyl group which is acylated to form a transient acetylenzyme intermediate during the hydrolysis of acetylcholine (Rosenberry, 1975). In addition to carboxylic acid esters, several other classes of acylating agents show reactivity towards the enzyme esteratic site. These include esters and acyl halides of substituted phosphonic, phosphoric and carbamic acids. The acyl enzymes formed by these compounds are hydrolysed at very slow, but measurable rates. Organophosphate-inhibited AChE may be rapidly dephosphorylated (or dephosphonylated) with nucleophilic agents, e.g. oximes, with concomitant reactivation of the enzyme (Rosenberry, 1975). In some cases, the amount of phosphonylated AChE which can be reactivated by oximes decreases as a function of time. This is due to acid-catalysed hydrolysis of a P-O-alkyl group on the enzyme to P-OH. Once dealkylation occurs (a process known as ageing) reactivation by oximes no longer occurs (Crone, 1974). 559

560

R.M. DAWSON and M. PorlilSKI

effects of this substance on AChE have been reported to date. In fact, Roufogalis and Wickson (1975) claimed that procaine had little or no affinity for the peripheral anionic site of ACHE. Nevertheless other workers have reported cooperativity between procaine binding sites on AChE (Foidart and Gridelet, 1974: Milyutin et al., 1977). Cooperativity is usually taken to indicate the existence of interacting polymeric forms of an enzyme or protein (Cornish-Bowden, 1976). Many allosteric enzymes are also cooperalive and vice versa: however, the terms are not interchangeable. They describe two different properties and should be clearly distinguished (Cornish-Bowden, 1976). In the case of ACHE, its allosteric properties have been well established (Rosenberry, 1975) but reports of cooperative behaviour are rare. It seemed worthwile, therefore, to p u r s u e the effects of procaine on AChE with particular emphasis on cooperative behaviot, r and some of the properties which are subject to manipulation by allosteric effectors, namely decarbamylation, ageing and the maximum velocity of substrate hydrolysis. The results are presented in this paper. Other work described below relates to the location of procaine binding sites on ACHE, as inferred from kinetic competition experiments between procaine and edrophonium, and between procaine and decamethonium. E d r o p h o n i u m is assumed to bind to the t r i m e t h y l a m m o n i u m binding subsite of the active catalytic centre of AChE (Taylor et aL, 19741 while the bisquaternary a m m o n i u m c o m p o u n d decamethonium binds by a two-site attachment. One site is the same as that which binds e d r o p h o n i u m and acetylcholine and the other is a peripheral site (Rosenberry, 1975: Berman et al., 1981). Before performing the kinetic experiments, it was necessary to establish that procaine is not hydrolysed by ACHE. Procaine is a substrate for serum cholinesterase (BuChE. acylcholine acylhydrolase: EC 3.1.1.8) (Kalow and Maykut, 1956; H a z a r d et al., 1967: Valentino et al., 1981) and could conceivably be a substrate for A C h E also. Kalow and M a y k u t (1956) reported a slight hydrolysis of procaine by A C h E of guinea-pig brain, a l t h o u g h the effect disappeared when care was taken to eliminate blood from the preparation. This suggests that the hydrolysis was due to the serum contaminant. Most experiments reported below were performed using a commercial preparation of solubilized bovine erythrocyte ACHE. Some experiments were also performed on a preparation of m e m b r a n e - b o u n d h u m a n erythrocyte AChE in order to resemble more closely the situation in t#ro. Allosteric properties of AChE

can vary markedly depending on the source of the enzyme (Dawson et al., 1981a). EXPERIMENTAL PROCEDURES Materials

Procaine hydrochloride was obtained from K & K Laboratories, Plainview and Hollywood. U.S.A. It was recrystallised from ethanol to constant melting-point (156 8'~C: lit. 154 6 C ; Clarke. 1969) and its identity and purity were checked by u.v. spectroscopy and thin-layer chromatography (Clarke, 1969). 3,3-Dimethyl-l-butanol was prepared in these laboratories (Dawson and Crone, 1975): no impurities were detected by gas liquid chromatography or ~H-NMR spectroscopy. Bovine erythrocyte ACHE, electric eel ACHE, horse serum BuChE and choline chloride were obtained from Sigma Chemical Co., and decamethonium iodide from Koch-Light. Edrophonium chloride was a gift from Roche Pty. Ltd. Membrane-bound human erythrocyte AChE was prepared as described previously (Dawson et al., 1981al. Neat human serum was used as the source of human serum cholinesterase. Methods Procedures for studying decarbamylation of dimelhylcarbamyl-AChE (Dawson. 1978), hydrolysis of acetylthiocholine [Dawson and Bladen, 19791 and ageing of isopropylmethylphosphonyl-AChE (Dawson and Bladen, 1979: Dawson et al., 1981a) have been described. Briefly, for decarbamylation. AChE was inhibited with neostigmine (60nMl and diluted extensively into buffer. At various times, afiquots of the solution were assayed tbr activity towards 0.5 mM acetylthiochofine. Ageing was studied by determining the ability of 0.5 mM N-methylpyridinium 2-aldoxime iodide to reactivate isopropylmethylphosphonyl-AChE as a function of time. The inhibited enzyme was prepared by incubating AChE in 5 mM Tris pH 8.7 with 50nM Sarim followed by removal of excess Sarin by chromatography. All experiments were performed in 2 mM KH.,PO4 that had been adjusted to pH 7.0 with 0~05 M NaOH. The temperature was 2 5 C for substrate hydrolysis and decarbamylation or 37 15' for ageing. In some experiments the buffer included 150 mM NaCI. Enzymic hydrolysis of procaine (20 or 40/~M) was studied i n 2 m M T r i s 150mM N a C l p H S . 0 a t 37'C by observing changes in the u.v. spectrum recorded with a Unicam SP800 spectrophotometer. Neat human serum (0.1 mB or a solution of horse serum BuChE (O.I ml) was added to 3.2 ml procaine solution. Hydrolysis of butyrylthioehofine land in some cases acetylthiocholine) was studied in 21raM Tris 150raM NaC1 pH 8.0 at 3T'C (Dawson and Btaden. 1979). Statistical tests of significance were determined using Student's t-test with P < 0.05 as a criterion of difference. Data are quoted as mean +_ standard error of the mean, unless stated otherwise, with the number of experi/nents in parentheses.

RESULTS Hydrolysis q[" hutyryhhiocholine

hv serum cholinester-

(IS~'S

The hydrolysis of butyrylthiocholine (BuSCh) fol-

Allosteric-like effects of procaine on AChE lowed Michaelis-Menten kinetics (equation 1) in the concentration range 0.2-2.0 mM. v =

-

-

v0

(1)

1 +Km/S

In equation (1), v represents the velocity of hydrolysis at substrate concentration s, and V0 (maximum velocity of hydrolysis) and Km (Michaelis constant) are constants. In duplicate experiments, K m was found to be 0.11 + 0.01 m M for human serum and 0.80 ___ 0.07 m M for horse serum cholinesterase.

Hydrolysis of procaine by serum cholinesterases A progressive change in the spectrum of 4 0 p M procaine was observed at pH 8.0, 37°C on addition of horse serum cholinesterase; see Fig. 1. The spectrum was practically unchanged over 5 h under the same conditions in the absence of enzyme, or in the presence of high active concentrations of AChE from electric eel, bovine erythrocytes or human erythrocytes (membrane-bound or Triton-solubilized). Similar results to those depicted in Fig. 1 were obtained when human serum replaced horse serum BuChE. The rate of enzyme-catalyzed hydrolysis of procaine was measured by following the rate of decrease of absorbance at 305 nm (Valentino et al., 1981). Human serum hydrolysed 4 0 p M procaine at a rate of 7.0 + 0.4 (5) × 1 0 - 7 M min-1. Practically the same rate of hydrolysis was observed for 20 pM procaine, which indicates that the value of Km for this system is much lower than 20 pM. On the other hand, horse serum BuChE hydrolysed 40 and 20ktM procaine at rates of 6.2 +__0.1 (5) × 1 0 - T M m i n 1 and 3 . 9 + 0 . 1 ( 5 ) x 1 0 - 7 M min -1, respectively. The value of K m calculated from these results (by manipulation of equation 1)

p -Aminobenzoic

ocid

Procoine

rnin

oo

I 200

250

Wavelength

300

nm

Fig. 1. Hydrolysis of 40 #M procaine by horse serum. The buffer was 2mM Tris-150 mM NaCI pH 8.0 at 37°C, and the reference cell contained serum and buffer. The spectrum was recorded immediately after addition of enzyme, and every I0 rain thereafter, to 60 min. The progression of hydrolysis is indicated by decreasing absorbance at 274325 nm and increasing absorbance at 240-274nm. The broken line represents the spectrum of 40/~M p-aminobenzoic acid (to which procaine is hydrolysed).

561

is 57.5 #M. Thus Km for procaine is much higher for horse serum BuChE than for human serum BuChE, in accordance with the K m values for BuSCh as substrate for these two enzymes (see above). At the same time as the hydrolysis of procaine was determined, dilutions of the serum samples were assayed in duplicate for their activity towards 2 mM BuSCh, under the same experimental conditions. By using equation (1), it was possible to calculate V0 for each enzyme-substrate combination. In the case of human serum BuChE-procaine, it was assumed that V0 equalled the observed velocity at 40 # M procaine. The ratio of V0 (BuSCh) to V0 (procaine), normalised to the same amount of serum enzyme, was thereby calculated to be 300 for human serum BuChE and 1575 for horse serum BuChE.

Effect of procaine on AChE-catalyzed hydrolysis of ASCh The rate of hydrolysis (v) of ASCh by AChE was studied as a function of ASCh concentration (s) at pH 7.0, 25°C in the presence of various concentrations of procaine (I). The symbol I is used since procaine is an inhibitor of hydrolysis. The results were analysed in several different ways. (i) A suitable concentration of procaine might induce cooperativity in binding of the substrate ASCh to ACHE, in which case v might vary with s according to equation (2) (Hill equation; Cornish-Bowden, 1976).

Vos~ v-

K , , + s ~"

(2)

In equation (2), I/0, Km and s have the same meanings as in equation (1), and n is the Hill coefficient. Positive cooperativity is indicated by n > 1, negative cooperativity by n < 1 and the absence of cooperativity by n = 1. Equation (2) reduces to equation (1) when n = 1. In the present work, the value of n was determined by non-linear regression using the computer program of Vaughn, Neal and Anderson (1976). (ii) The substrate concentration s was fixed (at 50 #M) and the procaine concentration I varied, in order to determine whether binding of procaine to AChE is cooperative. The results were analysed by linear regression according to equation (3), an alternative form of the Hill equation (Cornish-Bowden, 1976).

(')

log ~

=logK+nlogI.

(3)

The variable Yis the ratio of the velocity of hydrolysis

562

R.M. DAWSON and M. PORI ISKI Table h Effect of procaine on the hydrol)sis of AS('h by ACht! Soluble bovine erythrocyte AChE Ionic Stren@th

Membrane-bound human erythrocyte AChE

4 mM

Hill coefficient (eqn. 2)

n

0.87 ± 0.13

(5)*

1.05 ± 0.13

(6)f

Hill coefficient (eqn. 3)

n

0.86 ± 0.09

(7)

0.96 ± 0.42

(4)

Deviates from linearity for procaine concn. > 20 ~M (Fig. 2)

Km/V o plot

Ki

(BM)

5.5

VI/V O Ionic Strength

± 3.8

Two straight lines, 0-0.8 ~M and 0.8-3 BM procaine, significantly different in slope (Fig. 3)

(5)

0.19 ± 0.17

(6)

1.04 ± 0.08

(5) *

1.24 ± 0.20

(6) f

154 mM

Hill coefficient (eqn. 2)

n

0.97 ± 0.25

(2)~

0.96 ± 0.09

(4) §

Hill coefficient (eqn. 3)

n

0.94 ± 0.06

(4)

0.92 ± 0.09

(5)

Km/V o plot Ki VI/V O

(BM)

Linear,

0-400

99 ±

BM procaine 43 (5)

< I, decreasing with increasing procaine concn.

Linear, 12.3

0-80 ~M procaine ~ 2.2

(6)

Distributed about 1.0, showing no correlation with procaine concn.

Experiments were performed in 2 mM phosphate pH 7.0. 25 C, with or without 150 mM NaCI. Data are quoted as mean or calculated value ±95". confidence limits, with the number of experiments or linear regression points in parentheses. In determination of the Hill coefficient by equation 3, the concentration of ASCh was 50/lM (ionic strength 4 mM) or 200/tM (ionic strength 154 mM). * Procaine = 50/iM. + Procaine -- 3/~M. Procaine = 400 # M. § Procaine = 100 itM.

~' in the presence of procaine to that in its absence, and K is a constant. T h e value of n, the Hill coefficient, has the same implications as in (i) above. (iii) O n the a s s u m p t i o n that e q u a t i o n (1) adequately describes the kinetics of substrate hydrolysis in the presence of procaine (see below), K m and Vo were d e t e r m i n e d for various fixed c o n c e n t r a t i o n s of procaine ( D a w s o n a n d Bladen, 19791. Km/Vo was then plotted against I in order to d e t e r m i n e K~. the competitive dissociation c o n s t a n t for procaine, according to the linear equation (4), where (K~/Vo),~ is the ratio o f Km/Vo in the presence of procaine of c o n c e n t r a t i o n I to that in its absence ( D a w s o n and Bladen, 19791.

(K~,"Vot,d = I

+

I , K i.

(4)

The effect of procaine on the m a x i m u n l vclocit~ of hydrolysis V0 was evaluated quantitatively as the ratio Vl/l:'o where IPr and 1'o are the values of Vo in the presence and absence of procaine respectively. The various calculations described above are summarised in Table 1 which lists values of n, K~ and V,/Vo at two ionic strengths {4 and 154 mM} and for two sources of A C h E {soluble bovine erylhrocyte A C h E and m e m b r a n e - b o u n d h u m a n erythrocyte AChE}. Table 1 also includes c o m m e n t s on the linearity of the plot of K~.'Vo vs I {equation 4: see Figs 2

Allosteric-like effects of procaine on AChE

563

and 3). Only the initial linear portion of the plot was used in the calculation of K~. It is apparent from Table 1 that: (i) The Hill coefficient n was statistically indistinguishable from unity in all cases except for bovine erythrocyte AChE at low ionic strength at fixed ASCh concentration and varying procaine concentration (equation 3). Even in this case, the value of n (0.86) was close to unity, and it would be unwise to place too much emphasis on the statistical analysis in the light of the other determinations of n. (ii) Procaine significantly increased V0 for ASCh only for membrane-bound AChE at low ionic strength, in which case VI/Vo = 1.24, i.e. a 24% increase in the maximum velocity of hydrolysis was observed. In the other three cases (soluble bovine erythrocyte AChE at low ionic strength and soluble and membrane-bound AChE at the higher ionic strength) the ratio VI/Vo was less than or indistinguishable from unity, i.e. there was no increase in the maximum velocity of hydrolysis. (iii) Plots of K,,/Vo vs I were non-linear above a certain concentration of procaine at low ionic strength. (iv) The value of K~, the competitive dissociation constant for procaine, varied markedly with the enzyme source, and with ionic strength. The increase in K~ as the ionic strength was increased from 4 to 154mM was considerably higher for membrane-

"~E ~c

I

o

[

I

I

i

2

3

Procaine

concn

(/zM)

Fig. 3. Inhibition of membrane-bound human erythrocyte AChE by procaine. The buffer was 2mM phosphate pH 7.0, 25cC. Substrate = acetylthiocholine.

bound AChE (66-fold increase) than for soluble AChE (18-fold increase).

Inhibition of ASCh hydrolysis by two reversible inhibitors

IO

0

5

I

O

I IO Procoine

I 20

I conen

I

I ,50

(/a.M)

Fig. 2. Inhibition of soluble bovine erythrocyte AChE by procaine. The buffer was 2 mM phosphate pH 7.0, 25'~C. Substrate = acetylthiocholine.

All experiments were done in the absence of NaCI using bovine erythrocyte ACHE. The hydrolysis of 50/~M ASCh was studied in the presence of procaine and edrophonium, either alone or together. In the discussion below, Vo is the velocity of hydrolysis in the absence of inhibitors, vl is the velocity in the presence of edrophonium of concentration 11 (0.2-0.8/~M), v2 is the velocity in the presence of procaine of concentration I2 ((~20/~M) and v12 is the velocity in the presence of both edrophonium and procaine. In preliminary experiments, a plot of K~/Vo vs 11 (0.11 pM) was found to be linear and linear regression yielded K~ = 0.12 ~LM. Plots of Vo/V12 vs 11 at various fixed concentrations of procaine, and of Vo/Vl2 vs I2 at various fixed concentrations of edrophonium were also linear. However the plots of Vo/V~2vs I2 deviated from linearity if the concentration of procaine was increased above 20 pM, particularly in the absence of edrophonium, in accordance with the Km/Vo vs l plot for procaine (Fig. 2). The data from the double inhibi-

564

R.M. DAWSONand M. PORETSKI

r \ E

.2 •~

0.4

-

"

k

.

o hi

0

I0

Procoine

A

I

20

30

concn

(/~M)

Fig. 4. Isobologram for the inhibition of soluble bovine erythrocyte AChE by mixtures of edrophonium and procaine. The buffer was 2 mM phosphate pH 7.0, 25°C. Substrate = 50pM acetylthiocholine. Vo/Vl2= 1.30, 1.50, 1.83 and 2.78 for ©, O, A, • respectively.

tion experiments, keeping the concentration of procaine < 2 0 # M , were used to construct an isobologram, shown in Fig. 4. Isobols are equieffective combinations of active substances plotted on graphs whose coordinates are the concentrations of the substances. In the present case, the coordinates are 11 and I2 and the isobols correspond to combinations of the inhibi-

tors yielding a constant Vo/V,2 value at fixed concentration of ASCh (Fajszi, 1974). It is apparent from Fig. 4 that the isobols are parallel straight lines, which indicates that the binding of edrophonium and procaine to AChE is mutually exclusive, i.e. the two inhibitors compete for the binding site(s) (Fajszi, 1974). Double inhibition experiments in which procaine and decamethonium were the two inhibitors were performed at only one concentration of procaine (50 ttM). The concentration of decamethonium which reduced the rate of hydrolysis of ASCh by half (lso) was determined in the absence or presence of 50 #M procaine by analysis of plots of Vo/Vl vs I1 or Vo/Vl2 vs I1 respectively. The subscript 1 refers to the inhibitor decamethonium in this case. Such 15o values were determined at two concentrations of ASCh, 50/~M and 2.5 mM. Similar experiments were performed with 5 0 # M gallamine replacing 50/tM procaine. The results are set out in Table 2 in which it is apparent that both procaine and gallamine antagonized the inhibition caused by decamethonium, particularly at the lower substrate concentration.

Decarbamylation of dimethylcarbamyl-A ChE At low ionic strength (no NaCI present) the presence of procaine resulted in an increase in the rate constant (k) for decarbamylation, with bovine erythrocyte AChE as the enzyme source. The value of k r varied hyperbolically with the concentration of procaine (25-500 #M) and the results were therefore analysed as described previously for other accelerators of decarbamylation (Dawson, 1978), after subtracting the rate constant for spontaneous decarbamylation (k/) from the observed rate constants (k). The analysis gave a dissociation constant for the procaine-dimethylcarbamyl-AChE complex (Ka) of 50/~M and a cal-

Table 2. Antagonism of the inhibitory effects of decamethonium by procaine and gallamine Antagonist

Concentration of ASCh (mM)

150(A) (I,M)

I50(C) (lJM)

150(A)/I50(C)

Procaine

2.5

20.8

17.0

1.2

Gallamine

2.5

27.9

16.2

1.7

Procaine

0.05

5.~

0.83

6.0

Gallamine

0.05

12.3

0.79

15.6

Assays were performed in 2 mM phosphate pH 7.0 at 25°C. I50 = concentration of decamethonium which reduces the rate of enzymic hydrolysis of ASCh by half. 15o(A) = 150 in presence of antagonist. I5o(C) = 150 in absence of antagonist (i.e. control).

Allosteric-like effects of procaine on AChE

565

Table 3. Rate constants for decarbamylation of dimethylcarbamyl-AChE No NaCl

150 mM NaCl

S o l u b l e bovine e r ~ t h r o c ~ t e AChE Control

0.084

i

50 ~4 Procaine

0.211

± 0.010

0.002

(2)

20 J 4 DNB*

0.562

¢ 0.053

(5) t

50 ~M Procaine-20 ~4 DNB

1.145

~ 0.064

(5) t

500 ~M Procaine

Membrane-bound

0.315

t

0.007 ( 3 )

(4) t

0.339 ~ 0.025 (3)

human er~throcyte AChE

Control

0.338 ± 0.014 (4]

500 ~N Procaine

0.301 ± 0.012 (4)

Experiments were performed in 2 mM phosphate pH 7.0, 25°C, with or without added NaCI. Rate constants are given in units of h - I as mean + standard error of the mean, with the number of replicate experiments in parentheses. * DMB = 3,3-dimethyl-l-butanol. "t Statistically different from control, P < 0.05.

culated m a x i m u m rate constant k, (max) = 0.334 h - 1. The latter figure corresponds to an acceleration factor of 4.0. The alcohol 3,3-dimethyl-l-butanol (DMB), at a concentration of 20 mM, also increased the rate of decarbamylation, as reported previously (Dawson, 1978); see Table 3. W h e n decarbamylation was studied in the presence of 50/~M procaine and 20 m M D M B together, it was found that the c o m b i n a t i o n was synergistic, i.e. the rate constant for the combination exceeded the sum of the individual rate constants in a m a n n e r which was statistically significant (Lark, Craven and Bosworth, 1968). The actual values

observed are listed in Table 3. Table 3 also summarises the results of experiments at physiological ionic strength ( 1 5 0 m M NaC1). In this case procaine at a concentration of 0.5 m M (10 times K a at low ionic strength) was unable to influence significantly the rate of decarbamylation for soluble bovine erythrocyte A C h E or m e m b r a n e - b o u n d h u m a n erythrocyte ACHE.

Ageing of isopropylmethylphosphonyl-AChE First-order rate constants for ageing at pH 7.0, 37°C are listed in Table 4. F o r bovine erythrocyte

Table 4. Rate constants for ageing of isopropylmethylphosphonyl-AChE No NaCl

150 mM NaCI

Soluble bovine erythroc~te AChE Control

0.394 ± 0.006

(3)

0.282 ± 0.007

(3)

500 ~M Procaine

0.279 ± 0.011

(3)*

0.252

(3)*

± 0.006

Membrane-bound huaan erythrocyte AChE Control

0.377 % 0.025 (3)

500 ~M Procaine

0.334 ~ 0.016 (3)

Experiments were performed in 2 mM phosphate pH 7.0, 37°C, with or without added NaC1. Rate constants are given in units of h - I as mean + standard error of the mean, with the number of replicate experiments in parentheses. * Statistically different from control, P < 0.05.

566

R.M. DAWSONand M. PORETSK!

AChE in the absence of NaCI, the rate of ageing was significantly slower (by 29~o) in the presence of 0.5 mM procaine. A slight decrease in rate (11~o) was also observed in the presence of 0.5 mM procaine in 150 mM NaCI, and this decrease was just significant at the 95~o confidence level. Procaine (0.5 mM) also reduced the rate of ageing of membrane-bound human erythrocyte AChE in 150mM NaCI by 11~o, but in this case the decrease was not statistically significant. DISCUSSION The hydrolysis of procaine by human serum and horse serum cholinesterase was anticipated by the results of several other workers (Kalow and Maykut, 1956; Hazard et al., 1967; Valentino et al., 1981). Hydrolysis is due to cholinesterase rather than to other esterases of serum (Valentino et al., 1981). The value of K m for procaine and human serum cholinesterase has been reported to be 5-7/~M at 24-25°C (Kalow and Maykut, 1956; Hazard et al., 1967; Valentino et al., 1981). Our observation that K m for this system at 37°C is well below 20/~M is consistent with these reports. Furthermore, the hydrolysis of procaine by serum cholinesterases was found to be 2-3 orders of magnitude slower than that of a good substrate, butyrylthiocholine. Similar comparative rates were reported by Hazard et al. (1967), who used acetylcholine rather than butyrylthiocholine. On the other hand, no hydrolysis of procaine by acetylcholinesterase was detected after extended periods with relatively high concentrations of active enzyme. It was calculated for bovine erythrocyte AChE and electric eel AChE that any enzymic hydrolysis of procaine by either of these enzymes is at least 4-5 orders of magnitude slower than that of the good substrate acetylthiocholine. Procaine modified the catalytic properties of bovine erythrocyte AChE in a number of interesting ways. At low ionic strength it increased the rate of decarbamylation up to 4-fold, interacted synergistically with 3,3-dimethyl-l-butanol in the acceleration of decarbamylation (Table 3), reduced the rate of ageing (Table 4) and antagonised inhibition of ASCh hydrolysis by decamethonium (Table 2). These effects are typical of allosteric effectors of AChE (Changeux, 1966: Dawson et al., 1981a). Kinetic allosteric effects are generally not demonstrable at physiological ionic strength or require up to one thousand-fold higher concentrations of effector (Changeux, 1966; Rosenberry, 1975; Dawson et al., 1981b). The behaviour of procaine also followed this pattern; minimal effects of 0.5mM procaine on decarbamylation and ageing

were observed in 150 mM NaCI. On the other hand, some allosteric effectors of AChE are able to increase V0 for the hydrolysis of ASCh at low ionic strength (Dawson et al., 1981a) whereas procaine did not show this property (Table 1). Does procaine exert its effects by binding to a peripheral (allosteric) site on the enzyme at low ionic strength? We propose that at low concentrations (<20/~M) procaine binds only to the trimethylammonium binding site of the active catalytic centre, but that at higher concentrations it also binds to a peripheral, allosteric site. The evidence for this proposal is not conclusive, but we can cite the following points in its favour. 1. Procaine behaved as a competitive inhibitor of the hydrolysis of ASCh at low ionic strength if its concentration was kept at or below 20/~M. Within this concentration range it appeared to compete with the active-site inhibitor edrophonium. 2. At concentrations of procaine above 20 #M, inhibition of the hydrolysis of ASCh was more complex, as indicated by non-linearity of the K m / V o vs I plot (Fig. 2). This could be because of allosteric inhibitory effects being superimposed on pure competitive inhibition. 3. The allosteric-like effects of altered rates of decarbamylation and ageing required higher concentrations of procaine than those necessary for competitive inhibition of ASCh hydrolysis. For example the binding constant (Ka),for procaine in its acceleration of decarbamylation was found to be 50/~M. 4. Procaine at a concentration of 50/tM antagonised the inhibition of ASCh hydrolysis caused by decamethonium (Table 2), presumably by interfering with the binding of one of the quaternary ammonium groups of decamethonium to a peripheral anionic site. Changeux (1966) found that 50/~M gallamine antagonised inhibition of enzymic hydrolysis of 2.5 mM acetylcholine by decamethonium and cited this behaviour as evidence for the existence of peripheral anionic sites on AChE from Torpedo marmorata. 5. Changeux (1966) showed that the monoquaternary ammonium analogue of gallamine, 2512CT (Ph O CH2CHz-I~ (C2H5)3) had the same allosteric properties as gallamine itself, and therefore presumably bound to the same allosteric site, although the effects observed were much smaller in magnitude. Procaine (p-NH z- P h - C O - O CHzCH2 NH(C2Hs)z) has a number of structural features in common with 2512CT, and could well be accommodated by the same allosteric site. With regard to point 4 above, Changeux reported that the ratio of I5o for decamethonium in the pres-

Allosteric-like effects of procaine on AChE ence of gallamine to 15o in the absence of gallamine was 64 for AChE from T. marmorata. In the present work with bovine erythrocyte ACHE, the same ratio with the same concentrations of gallamine (50pM) and substrate (2.5 mM), but with ASCh as the substrate instead of acetylcholine, was found to be only 1.7 (Table 2). A higher ratio (15.8) was observed at a lower substrate concentration (0.05 mM). These relatively low values are not surprising in the light of the report by Roufogalis and Quist (1972) that decamethonium and gallamine do not bind to a common peripheral site of bovine erythrocyte ACHE. Changeux (1966) presented other evidence for the existence of allosteric sites on T. marmorata ACHE, but similar studies on the interaction of procaine with bovine erythrocyte AChE are not as informative. For example, Changeux found that gallamine could not completely inhibit the enzymic hydrolysis of acetylcholine, and a residual rate of hydrolysis remained which was unaffected by increasing concentrations of gallamine. In the present work, greater than 95~0 inhibition of the enzymic hydrolysis of ASCh could be achieved with a sufficiently high concentration of procaine. This lack of a plateau effect does not prove or disprove the existence of allosteric sites. Also, Changeux reported that gallamine increased the ability of the active-site inhibitor m-(trimethylammonium) phenol to inhibit the enzymic hydrolysis of acetylcholine, whereas in the present work inhibition of the enzymic hydrolysis of ASCh by the closely related compound edrophonium (m-(dimethylet hylammonium)phenol) and procaine was purely additive and consistent with kinetic competition at the same site (see above). Kitz, Braswell and Ginsburg (1970) also provided evidence for the existence of peripheral sites on ACHE, this time from the electric eel, Electrophorus electricus. Their conclusions were based on the effects of gallamine on decarbamylation, but differences in the properties of eel AChE and bovine erythrocyte AChE prevent the use of their criteria in the present case. For example, decarbamylation was faster for eel AChE in the presence of gallamine and choline (which is presumed to bind to the trimethylammonium site of the active catalytic centre) than in the presence of either compound alone, which suggests separate binding sites for the two compounds. However rates of decarbamylation for gallamine and choline are not additive in the case of bovine erythrocyte AChE (Dawson, 1978). Also, Kitz et al. (1970) reported that the active-site inhibitor m-(trimethylammonium)phenol inhibited decarbamylation and that this effect was antagonised by gallamine in a manner not consistent with competition at the same binding site. In the

567

present work, however, the analogous inhibitor edrophonium did not inhibit decarbamylation at concentrations up to 10 -5 M, nearly 100 times higher than the competitive inhibition constant with respect to hydrolysis of ASCh. It was therefore not possible to determine whether procaine could antagonise inhibition of decarbamylation. If procaine does perturb rates of decarbamylation and ageing by binding to a peripheral site, then this site must be different from the peripheral anionic site to which Ca 2÷ binds with consequent acceleration of substrate hydrolysis, since Roufogalis and Wickson (1975) reported that procaine has little or no affinity for the latter site. This observation has little bearing on the likelihood of the existence of other peripheral binding sites for procaine, because the allosteric effector gallamine apparently does not bind to the peripheral calcium-binding site either (Roufogalis and Quist, 1972). Studies to define further the site of action of various allosteric effectors and procaine by use of protein-modifying reagents (O'Brien and Test, 1978) are currently in progress. Despite its allosteric-like properties there was little evidence for any cooperative behaviour at low or physiological ionic strength in the binding of procaine to the enzyme or in the binding of the substrate acetylthiocholine in the presence of procaine (Table 1). The results therefore do not substantiate two reported claims of cooperative behaviour; Milyutin et al. (1977) reported negative cooperation between procaine binding sites for rat brain AChE while Foidart and Gridelet (1974) reported a Hill coefficient n = 1.7 (i.e. positive cooperativity) for the inhibition by procaine of the hydrolysis of acetylcholine by membrane-bound Torpedo ACHE. Most of the experiments performed in this study were done using soluble bovine erythrocyte ACHE. Some experiments were also performed with membrane-bound human erythrocyte AChE for comparison, and because allosteric effects can sometimes be observed at physiological ionic strength for membrane-bound AChE where effects are absent under the same conditions for soluble AChE (Crone, 1974; Dawson et al., 1981a). In the present work, however, 0.5mM procaine failed to influence the rates of decarbamylation and ageing for membrane-bound AChE at physiological ionic strength (Tables 3 and 4). No cooperative behaviour was induced by procaine either (Table 1), in agreement with the results for soluble ACHE. On the other hand, the maximum velocity V0 for the hydrolysis of acetylthiocholine by membrane-bound AChE was significantly increased by procaine at low ionic strength (Table 1), in con-

568

R. M, DAWSON and M. PORETSKI

trast with the situation for soluble ACHE. Other differences between the two forms of the enzyme were also apparent when studying the effect of procaine on substrate hydrolysis: procaine was a much more powerful inhibitor for m e m b r a n e - b o u n d AChE than for soluble AChE at low ionic strength and its inhibitory power decreased more dramatically for membrane-bound AChE than for soluble AChE as the ionic strength increased (Table 1). The main conclusion from this paper therefore is that. despite the uncertainties as to its site(s) of action, procaine may well be added to the list (Rosenberry, 1975; Dawson, 1978) of allosteric effectors of AChE at low ionic strength. The significance of this conclusion lies in two areas. Firstly, the c o m p o u n d s which are known allosteric effectors of ACHE, e.g. d-tubocurarine and gallamine, are pachycurares, i.e. they block depolarization induced by acetylcholine at the neuromuscular junction by binding to the nicotinic acetylcholine receptor (Changeux, 1966; Tsai et al., 1979). According to recent publications, tubocurarinc and gallamine also bind to the ionic channel associated with the receptor (Colquhoun and Sheridan, 1981: Shaker et al., 1982). Procaine is the first allosteric effector of AChE to lack a significant receptor-binding capacity. Procaine does block depolarization, but it does so primarily by its interaction with the ionic channel (Tsai et al., 1979). Procaine also interacts with the muscarinic acetylcholine receptor at an accessory site other than the true receptor site (Aguilar et al., 1980). Secondly, the results give a new dimension to the spectrum of cholinergic actions of procaine modifier of the ionic channel receptor complex (above), substrate for serum cholinesterase (see lntroduction), inhibitor of AChE (Wills, 1963: Foidart and Gridelet, 1974), acetylcholine agonist in snail neuron (Israel and Meunier, 1979k- and now, allosteric effector of ACHE. O n the other hand, no evidence of cooperativity in the kinetics of AChE was found in the presence of procaine, contrary to two reports in the literature of experiments on other sources of the enzyme.

REFERENCES Aguilar, J. S., Criado, M. and De Robertis, E. (1980). Inhibition by local anesthetics, phentolamine and propranolol of [3H]quinuclidinyl benzylate binding to central muscarinic receptors. Eur. J. Pharmac. 68, 317 326. Berman, H. A., Becktel, W. and Taylor, P. (1981). Spectroscopic studies on acetylcholinesterase: Influence of peripheral-site occupation on active-center conformation. Biochemistry 20, 4803 4810. Bolger, M. B. and Taylor, P. (1979). Kinetics of association

between bisquaternary ligands and acetylcholinesterase. Evidence for two conformational states of the enzyme from stopped-flow measurements of fluorescence. Biochemistry 18, 3622-3629. Changeux, J. P. (1966). Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Molec. Pharmac. 2, 369 392. Clarke. E, G. C. (1969). Isolation and Identification o! Druos. The Pharmaceutical Press, London. Colquhoun, D. and Sheridan, R. E. (1981). The modes of action of gallamine. Proc. R. Soc. Lond. B. 211, 181 203. Cornish-Bowden, A. J. (1976). Principles of Enzyme Kinetics. Chap. 7. Butterworths, London. Crone, H. D (19741. Can allosteric effectors of acetylcholinesterase control the rate of ageing of the phosphonylated enzyme'? Biochem. Pharmac. 23, 460-463. Dawson, R. M. (1978). Carbamylation and decarbamylation of acetylcholinesterase: effect of choline, 3.3-dimethyl-lbutanol and some allosteric effectors. J. Neurochem. 30, 865 870. Dawson. R. M. and Bladen, M. P. (1979I. Some adjuncts to oxime-atropine therapy for organophosphate intoxication their effects on acetylcholinesterase. Biochem. Pharmac. 28, 2211 2214. Dawson, R. M. and Crone, H, D. 11975). The effect of aliphatic alcohols on the hydrolysis of acetylcholine by acetylcholinesterase, d. Neurochem. 24, 411 414. Dawson. R. M., Crone, H. D.. Bladen, M. P. and Poretski, M. (1981ak A comparison of the effects of ionic strength on three preparations of acetylcholinesterase in the presence and absence of gallamine. Neurochem. Int. 3, 335 341. Dawson, R. M., Poretski, M. and Upsher, C. M. (1981b). Gallamine and tubocurarine as possible allosteric modifiers of soluble acetylcholinesterase activity at physiological ionic strength. Neurochem. Ira. 3, 405 409. Fajszi, C. (1974). Methods of analysis of double inhibition experiments. Syrup. Biol. Hun q. Ig, 77 103. Foidart, J. M. and Gridelet. J. (1974). Effects of procaine and d-tubocurarine on the activity of membrane-bound acetylcholinesterase. Bioehem. Pharmac. 23, 725 733, Hazard, R., Rodallec, A. and Larno, S. (1967). Serum cholinesterases of human and equine origin have the same hydrolyzing effect upon procaine as the corresponding serum .I. Physiol., Paris 59, 9 16. Israel. J. M. and Meunier, J. M. (1979). Procaine as an acetylcholine agonist in snail neuron. J. Pharmae. exp. Ther. 211, 93 98. Kalow, W. and Maykut, M. O. (1956). The interaction between cholinesterase and a series of local anesthetics. J. Pharmac. ex'p. Ther. 116, 418 432. Kitz. R. J., Braswell, L. M. and Ginsburg, S. (1970). On the question: Is acetylcholinesterase an allosteric protein? Molec. Pharmac. 6, 108 121. Lark, P. D., Craven, B. R. and Bosworth, R. C. L. tl968). The Handlinq o{Chemical Data, p. 129. Pergamon Press, Oxford. Milyutin, A. A., Okun, I. M., Aksentsev, S. L., Arinchin, N. i. and Konev, S. V. (1977). Age-related changes in the allosteric properties of brain acetylcholinesterase. Chem. Ah~s. 86, 53141. O'Brien, R. D. and Test, K. E. (1978). Exploration of the binding sites of acetylcholinesterase with protein-modifying reagents. Arch. Biochem. Biophys. 187, 113 120.

Allosteric-like effects of procaine on AChE Rosenberry, T. L. (1975). Acetylcholinesterase. In Advances in Enzymology, (Meister, A. ed.) Vol. 43, pp. 110-111, 190-196. John Wiley and Sons, New York. Roufogalis, B. D. and Quist, E. E. (1972). Relative binding sites of pharmacologically active ligands on bovine erythrocyte acetylcholinesterase. Molec. Pharmac. 8, 41-49. Roufogalis, B. D. and Wickson, V. M. (1975). Acetylcholinesterase: Specificity of the peripheral anionic site for cholinergic ligands. Molec. Pharmac. 11, 352-360. Shaker, N., Eldefrawi, A. T., Aguayo, L. G., Warnick, J. E., Albuquerque, E. X. and Eldefrawi, M. T. (1982). Interactions of d-tubocurarine with the nicotinic acetylcholine receptor/channel molecule. J. Pharmac. exp. Ther. 220, 172-177. Taylor, P., Lwebuga-Mukasa, J., Lappi, S. and Rademacher, J. (1974). Propidium--a fluorescent probe for a peripheral anionic site on acetylcholinesterase. Molec. Pharmac. 10, 703 708.

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Tomlinson, G., Mutus, B. and McLennan, I. (1980). Modulation of acetylcholinesterase activity by peripheral site ligands. Molec. Pharmac. 18, 33-39. Tsai, M., Oliveira, A. C., Albuquerque, E. X., Eldefrawi, M. E. and Eldefrawi, A. T. (1979). Mode of action of quinacrine on the acetylcholine receptor ionic channel complex. Molec. Pharmac. 16~ 382-392. Valentino, R. J., Lockridge, O., Ekerson, H. W. and La Du, B. N. (1981). Prediction of drug sensitivity in individuals with atypical serum cholinesterase based on in vitro biochemical studies. Biochem. Pharmac. 30, 1643 1649. Vaughn, W. K.. Neal, R. A. and Anderson, A. J. (1976). Computer estimation of the parameters of the sigmoidal kinetic model. Comput. Biol. Med. 6, 1 7. Wills, J. H. (1963). Pharmacological antagonists of the anticholinesterase agents. In Handbuch der Experimentallen Pharmakologie (Koelle, G. B., ed.), Vol. 15, p. 895. Springer, Berlin.