Kinetic studies on the acetylcholinesterase (AChE) from Allolobophora caliginosa

Comp. Biochem. Ph~,iol.. Vol. 62C. pp. 173 to 180 © Pergamon Press Ltd 1979. PrinTed in Great Britain

0306-4492 79 0301-0173S02.00 0

KINETIC STUDIES ON THE ACETYLCHOLINESTERASE (ACHE) FROM A L L O L O B O P H O R A C A L I G I N O S A GIOVANNI B. PRINCIPATO, M. VITTORIA AMBROSINI and ELVIO GIOVANNINI Institute of General Biology. Faculty of Medicine and Surgery, University of Perugia. Perugia. Italy

(Received 20 July 1978) A b s t r a c t - - l . Kinetics of the acetylcholinesterase (ACHE) extracted from Allolobophora caliginosa were

studied using acetylthiocholine (ATC) and butyrylthiocholine (BTC) as substrates: tetraethylammonium (TEA), tetrabutylammonium (TBA) and choline were used as inhibitors. 2. Competitive inhibition was observed using choline up to 10 mM; higher concentrations of choline as well as the presence of TEA or TBA always led to non-competitive effect. 3. A model of AChE kinetic was described, supposing the existence of a peripheral anionic site: the results obtained are in agreement with the equations resulting from such a model.

INTRODUCTION

During recent research we performed purification and partial characterization of the acetylcholinesterase (acetylcholine hydrolase E.C. 3.1.1.7; ACHE) extracted from Allolobophora caliginosa (Principato et al., 1978a). In order to obtain a further characterization of such an enzyme, we carried out a kinetic analysis using acetylthiocholine (ATC) and butyrylthiocholine (BTC) as substrates; t e t r a e t h y l a m m o n i u m (TEA), t e t r a b u t y l a m m o n i u m (TBA) and choline were used as inhibitors. For the purpose of interpreting the kinetic measurements we considered a reaction scheme derived from the one with a single binding site of K r u p k a & Laidler (1961). O u r scheme is shown in Fig. I; according to it, the inhibitor could join to the e n z y m e - s u b s t r a t e complex as well as to the free enzyme on a second anionic site. IES and IE are not catalytically active; they can only dissociate giving respectively ES and E. Deacylation occurs only for EA and EAI in which I is b o u n d to the anionic part of active site. The examined reaction scheme was devised in order to have the functions 1/vi vs I-S] and KJV~ vs [ I ] as straight lines. F o r this purpose, in the step

IES

k~ • IE Xk_6[S ]

we had to suppose k 6 = k _ 6 = 0. In fact, k - 6 ~: 0 condition would have made v~ function a seconddegree equation in [S-I, while k - 6 4 : 0 condition would have made KJV~ a parabola, as we previously observed (Principato et al., 1978b). The existence of a second peripheral binding site was proved in A C h E from several species of animals using various experimental approaches (Aldridge & Rainer, 1969; Hellenbrand & Krupka, 1970; Kato et al., 1972; Rosenberry & Bernhard, 1972; Mooser et al., 1972; Steinberg & Amshey, 1976). Moreover, the presence of such a second site in the A C h E of Inverteb r a t a in general has already been suggested (Hellenb r a n d & Krupka, 1970).

MATERIALS AND METHOD~ Reagents AChE was purified from A. caliginosa by a method that has already been described (Principato et al.. 1978a). We used the native form of lowest molecular weight (180,000). The lyophilized material had a specific activity of 2029 mU per milligram of proteins. The stock solutions were prepared daily and stored at 4°C until use. Acetylthiocholine iodide (ATC). butyrylthiocholine iodide (BTC), tetraethylammonium iodide (TEA), tetrabutylammonium iodide (TBA). choline chloride and 5,5-dithiobis-2-nitrobenzoic acid (DTNB) used in the AChE activity measurements were purchased from Merck.

Kinetic measurements AChE activity was measured according to the method of Ellman et al. (1961) at 25°C room temperature in reaction mixture containing 50 mM phosphate buffer--pH 7.4, 100 mM NaCI, 10 mM DTNB and non-inhibiting substrate concentrations (ATC up to 0.75mM or BTC up to 25 mM). Inhibitor concentrations were respectively 0.3-3mM (TEA), 0.1-1.5mM (TBA), 3-50mM (choline), in order to obtain inhibitions of the same size. The product of thiocholine reaction with DTNB was determined on a Beckman DK-2A spectrophotometer. The absorbance at 405 nm was recorded as a function of time. The rate of change in absorbance was linear over a period of 2 min for all concentrations of substrate and the slope was used to evaluate the initial velocity. All measurements were carried out in 100mM NaCI, although activity of our AChE seemed to be scarcely affected by low ionic strength, unlike bovine erythrocyte AChE (D6sir6 et al., 1973). Evaluation of the constants was performed by the method of Wilkinson (1961). Equation of velocity According to the model shown in Fig. 1, the reciprocal of the initial velocity is: 1

1

1

kEEA] + ak[EAl] (1) [EA] and [EAI-I were obtained by King and Altman's method as reported by Plowman (1972): to use this method, one must arrange the mechanism into some convenient closed-loop type of geometric array: r,

VI 173

174

GIOVANNI B. PRINCIPATO. M. VITTORIA AMBROSINIand ELVIO GIOVANNINI

The next step is to write down all possible patterns with n - 1 lines (n = number of enzyme forms), in which all lines are connected but no closed loops occur:

S -VI-f -T -T U 3According to Plowman (1972) and by means of elementary calculations we obtained:

k

v,

['](1

l+k;+K-33\

y shows a m i n i m u m when ( B E - CD)<<. 0" this occurs when a > A. ,3. Equation (8) is negative in the other possible cases: a = 1 with k-2 < k : 0 < a < ~ w i t h k-2 > k : a > ~ with k_ 2 < k; k_ 2 = k. The function is always increasing and shows a downward concavity. The condition 0 < a < ~t with k - 2 < k is meaningless, because it would require a<0.

ak)( a[n rk[Z] k +kT+k-~ + l+~/lk'K4+k'[S]\ ak

,2,[

,{i [,]

+~+Ks/]

The function (Vo/Vi - 1)/[I] (3) is a hyperbola of the type

where

K.1 = (k-1 + k')/kt: K2 = k-2/k2:

v

K 3 = (k_ 3 + ak)/k3; g 4 = k_,,/k4; Ks = k_5/k5.

with A, B, C, D /> 0 and [1] >/0. In this case the signs of y' and y" derivatives are linked to that of the expression

(~_ l)/[i] = -K~[l - a(l - k~) ] + F kK' ( I +

1+

k

+

[I] ,/

/ 1

+#

K,

/

+ k'[S]/\

L) +&](' + aUl/

K3J

(3)

+ T~}

ak) k [ l ] ( 1 + a[/_~/ ak + ~_2 + + L" k'g,, \ K,3 / kEo(l

Ki

(it)

C[I] + D

F r o m equation (2) we have derived the following functions of I-I], according to D6sir6 & Blanchet (1975):

I

A[I] + B

(4)

a[l] I + K3/

[I]

(5)

+ Ks,/ where v0 is the velocity in the absence of inhibitor, 1/V~ is the ordinate intercept and KffV~ is the slope of Lineweaver and Burk plots in the presence of inhibitor. The functions (2) and (4) are definable as 2:1 type according to Cleland (1963) and can be represented by the following general equation: y =

A [ I ] 2 + B[I] + C

(6)

oU] + E

where 4, B, C, D, E are positive constants for all [S] values, while [I] can show only values >i 0. The possible shape of y depends on the sign of the second derivative (y"); such a sign is connected to that of the expression:

AE 2

-

D(BE - CD).

(7)

As regards l/vi (2) and 1/1/// (4), the expression (7) show the same following value

a(KE°~2[a( j l

l]

(8,

and its sign depends on the value of both a and k/k_2 ratio. Setting (1 - k/k_2}-1 = ~, we can distinguish three different cases. 1. Equation (8) is equal to zero when: a = 0; a = ct; a = 1 with k_ 2 > k" so (2) and (4) will be straight lines. 2. Equation (8) is positive only when a > a with k - 2 > k; (2) and (4) show an upward concavity, y is always increasing when (BE - CD) > 0; this occurs when a > A. As regards (2)

[ A =~

kg 3 kglga(l 1 +k~-4 + T~\~-2

I )] + ~-~5

(9)

and, as regards (4) A = 1+

kK3 k'K4 "

(10)

(AD

-

CB)" this is equal to

1+#+k, ES]/L \

~

-I

,

(t2)

We distinguish, also in this case, three different condi"tions. (i) W h e n a = 0, a = ct, a = 1 with k-2 > k, (12) is equal to zero and this implies that (3) is a constant. (ii) W h e n a > ct with k-2 > k (12) is positive and (3) is a hyperbola decreasing together with [I]. (iii) W h e n a = 1 with k_ 2 < k, 0 < a < ct with k_ 2 > k, a > ~ with k-2 < k, k_ 2 = k, (12) is negative and this fact implies that (3) is a hyperbola decreasing as [I] increases. The equation (5) represents a straight line with every value of a, k, k_ 2.

RESULTS

T h e results o f m a t h e m a t i c a l a n a l y s i s carried o u t o n o u r r e a c t i o n m o d e l with t w o b i n d i n g sites (Fig. 1) are r e p o r t e d in T a b l e 1. I n p a r t i c u l a r , it s h o w s t h e p o s s i b l e s h a p e s o f t h e v a r i o u s [ I ] f u n c t i o n s at different v a l u e s o f a, k, k_ 2 a n d t h e c o r r e s p o n d i n g s h a p e s in t h e m o d e l , with a single b i n d i n g site ( K r u p k a & Laidler, 1961). O u r a n a l y s i s h a d c o n s i s t e d o f c o m p a r ing e x p e r i m e n t a l data, p l o t t e d as [ I ] functions, with t h e t h e o r e t i c a l f u n c t i o n s s h o w n in T a b l e 1. T h e pres u m p t i v e linearity o f e x p e r i m e n t a l p l o t s w a s statistically verified as well as n o n - l i n e a r i t y o f t h e reciprocals, in o r d e r to d i s t i n g u i s h t h e 2:1 f u n c t i o n s f r o m h y p e r b o l a s . A n e x a m i n a t i o n o f t h e results d e m o n s t r a t e s t h a t t h e s h a p e s of [ I ] f u n c t i o n s c o n s i d e r e d by us are s i m i l a r with b o t h s u b s t r a t e s ( A T C a n d BTC).

Kinetic studies on the acetylcholinesterase k- 4

k 3 [I]

k r

'N.

IES

),

ES

175

EAI

EA

k 4 [I]

k- 3

k-5

k2

IE

[I] El

E ks

El]

k- 2

Fig. 1. Reaction scheme with two binding sites. mainly with ATC as a substrate; in fact, this subAs regards the various inhibition patterns, Dixon stance was used at concentrations m u c h lower than (1/V i VS [I]) relative to the three inhibitors, BTC. Because such a decrease in initial velocity does reported in Fig. 2, show that TEA and TBA inhibinot occur, we believe it correct to discard the a = 0 tions are in each case specifiable by a d o w n w a r d concondition. cavity curve; choline inhibition shows a straight-line In addition, examining the plots on the whole, there pattern. K J V ` function is a straight line in all conare no substantial differences between the patterns of sidered cases (Fig. 3). (co/ri - l ) / [ l ] function is a deTEA and TBA inhibitions: such an inhibition can be creasing hyperbola in the presence of TEA or TBA; on the contrary, in the presence of choline, it shows assigned to one of the following cases of our model constant values within the considered [I] range (Fig. 4). • (Fig. 1, Table 1): a = 1 with k-2 < k, 0 < a < ~ with : t w i t h k _ , < k,k__, = k. At the present In consequence of TEA and TBA inhibition, l/v` k_2 > k , a > function (Fig. 5) takes a d o w n w a r d curvature. O n the time we c a n n o t perform a further distinction a m o n g these four possibilities. contrary, in the presence of choline it shows constant values until 1 0 m M [ I ] ; when choline concentration DISCUSSION increases, l/v` function shows increasing values and AChE from A. caliyinosa shows inhibition patterns straight-line pattern. Considering the shape of ( t ' o / r i - 1)/[I] and l/V. with ATC and BTC that c a n n o t be explained by the functions (Figs 4 and 5), choline until 1 0 m M leads reaction model with a single bidding site: on the concompetitive inhibition corresponding to the a = 1 or trary, such patterns agree with the model suggested a = ~t cases of the model with a single binding site by us, that considers the existence of a peripheral site. The inhibitor might then join to several enzymatic of K r u p k a & Laidler (1961) (Table 1). The different forms, at least E and ES, without affecting the catashape of l/V,. function when choline concentration inlyl~ic mechanism of the active site. In fact, according creases demonstrates a change in the kinetic mechanto our experimental data, no difference is detectable ism: inhibition pattern is then consistent with the between inhibition patterns with ATC and BTC as cases a = 1 with k_ 2 ~ k , o ~ ~. (./ = 0 of our model substrates. AChE hydrolyses ATC faster than BTC (Fig. 1, Table I). The a = 0 condition is perhaps the and such a difference also remains in the presence least likely, because it would require the block of deof the various inhibitors. acetylation by choline. In this case the velocity should The appearance of non-competitive inhibition only tend to zero with high inhibitor concentrations,

plots

Table 1. Theoretical shape of the plots of the various I-I] functions at different values of the kinetic parameters Ca. k. k__,l according to the reaction scheme with two binding sites (Fig. 1) (Co/li - 1)/[I]

l /ti

[I] Functions 1/I,4

Ki/ l'~

Values of the kinetic parameters

Straight line (i.d.)

Const. (i.d.I

Straight line (const.)

Straight line (i.d.)

.a = 0 .a = 1 with k , > k . a = :t with k _ , > k

2:1 Upward concavity ll.d.)

Increasing hyperbola (i.d.)

2:1 Upward concavity (Decreasing hyperbolaj

Straight line (i.d.)

. a > 2 with k__,> k

2:1 Downward concavity (i.d.)

Decreasing hyperbola (i.d.)

2:1 Downward concavity (Increasing hyperbola)

Straight line

.a = .0 < .a > .k_,

(i.d.)

1 with k__, < k a < ~t with k_, > k :t with k - 2 < k = k

The corresponding shape of the same functions according to the scheme with a single binding site of Krupka & Laidler (1961) is reported in parentheses.

1

i

I [I]

mM

1.5 '

Choline

25

50

Fig. 2. Dixon plots (l/l,~ vs [I]) relative to inhibition of the AChE by tetraethylammonium (TEA), tetrabutylammonium (TBA) and choline in 50 m M phosphate buffer--pH 7.4:0.1 M NaCI; 0 . 5 m M acetylthiocholine (ATC O - - O ) or 25 m M butyrylthiocholine (BTC O--~-I)) were used as substrates, l/t'~ values are normalized with respect to 1/(,o (reciprocal of the initial velocity in the absence of inhibitors). Each point is the mean of 6 measurements.

,

1

1

I

1.0 '

S-

S"

5.

0'.5

lO-

,

TBA

lO-

TEA

lO,

v;

z z

< 3) Z

5

m r" <

::1 e'~

z

?,

,.q O

.<

p

,"b

z

© < >. Z

/

TEA

1

5"

10.

0.5

[I]

TBA

mM

1.0

, 1.5

1

lO

2o

3o-

4o-

25

Choline

50

Fig. 3. Plots of the Ki/E function vs tetraethylammonium (TEA). tetrabutylammonium (TBA) or choline concentrations, using 0.5 mM acetylthiocholine (ATC O O) or 25 mM butyrylthiocholine (BTC • - - • ) as substrates. Ki/|/i values are normalized with respect to Ko/I/o (slope of Lineweaver and Burk plots in the absence of inhibitors). Each point is the mean of 6 measurements.

lO-

20-

30.

v|

K_i

m

R

0

0

2'

3'

•

] ?

~ 5

Ii] m M

TBA

10 i

A 1.5 '

v

3

C~.

N

25

E)

Choline

O

50

C

1)/[I] function vs tetraethylammonium (TEA), tetrabutylammonium (TBA) or cholinc conccntr~ltions, using 0.5 mM acctylthiocholinc (ATC O O) or 25 m M butyrylthiocholine (BTC • •1 as substratcs. Each point is the mean of 6 measurements.

I'

A

0

5-

'SO"

5"

10-

,.t

Fig. 4. Plots. of the (Z.o/Z'i-

[,]

Vi

Yo

14-

TEA

7' Z

<

m

e..,

70

~r

.-] ©

.<

D

:-.)

z

< > "7

!

!

0 I

3-

6-

oi,

TBA

I] m M

,:o

i~

,.,

2-

3-

4-

Choline

2u8

5'0

Fig. 5. Plots of the I/V, function vs tetraethylammonium (TEA). tetrabutylam'monium (TBA) or choline concentrations, using 0.5 mM acetylthiocholinc (ATC O - - O ) or 2 5 m M butyrylthiocholine (BTC O-- e l as substrates. 1/1~ values are normalized with rcspcc! to 1/1.~> (ordinate intercept of Lincwcaver and Burk plots in the absence of inhibitorst. Each point is the mean of 6 measurements.

1

2,

$

TEA

@

g-

@

R"

180

GIOVANNI B. PRINCIPATO. M. VITTORIA AMBROSINIand ELVlO GIOVANNINI

for high choline concentrations may be due to a lower affinity for the peripheral site of the A C h E of A. caliginosa; the bond might thus become detectable only with high concentrations of inhibitor. O n the other hand, the involvement of such a site in the choline inhibition mechanism was also suggested by other authors as regards A C h E from bovine erythrocytes (D6sir~ et al., 1975). Differences between inhibition patterns of AChE from A. ca@inosa in the presence of TEA or TBA cannot be detected by the kinetic model used by us: however, b o t h TEA and TBA show the involvement of a peripheral anionic site in the enzyme activity mechanism according to the purposes of the present study. In addition, using TEA or TBA instead of choline, it is possible to put in evidence such a site with far lower concentrations ol inhibitor: the site seems to show a higher reactivity for TEA and TBA. O u r results concerning the existence of a peripheral site in the A C h E of A. ca@inosa agree with those obtained by various authors studying A C h E from other animal species (Aldridge & Reiner, 1969; Hellenbrand & Krupka, 1970: Kato et al., 1972; Rosenberry & Bernhard, 1972: Mooser et al., 1972; Steinberg & Amshey, 1976). However, our observations differ from other reports as regards TEA and TBA; in fact, such substances have long been k n o w n to show two different inhibition patterns, competitive (TEA) and non-competitive (TBA) respectively (Krupka, 1965), b o t h explainable by the model with a single binding site of K r u p k a & Laidler (1961). In addition, on the basis of more recent research carried out using bovine erythrocyte AChE (D6sir6 et al., 1975), inhibition mechanism of TEA and TBA agree with such a model. In particular, TBA inhibition seems to belong to the a < ct with k - 2 > k or a > ct with k - 2 < k cases; however, it is clearly different from TEA inhibition, described as a > ct with k_2 > k case. O n the contrary, a possible involvement of a peripheral anionic site was observed by the same authors only with T h i a z i n a m i u m and Thioridazine ions. Using TEA or TBA, the existence of a peripheral anionic site has not until now been demonstrated in A C h E from species other t h a n A. caliginosa; therefore, it is likely that this last has an anionic site more reactive against the considered inhibitors. The possible role of a peripheral site in the cholinergic system has not until now been elucidated (Mooser & Sigman, 1974); however, at least in vitro, it seems to play an i m p o r t a n t role in inhibition mechanisms of the AChE also in A. cali~tinosa. SUMMARY Authors carried out a kinetic study o n the acetylcholinesterase (ACHE) extracted from Allolobophora ealiginosa. During such a research acetylthiocholine (ATC) and butyrylthiocholine (BTC) were used as substrates; t e t r a e t h y l a m m o n i u m (TEA), tetrabutylamm o n i u m (TBA) and choline were used as inhibitors. Reaction scheme with a single binding site was not suitable to explain the results obtained: on the contrary, these agree with a kinetic model with two binding sites devised by the authors, thus proving the existence of a peripheral anionic site in the A C h E from A. caliginosa. No difference was seen between

the inhibition patterns in the presence of either substrate. Using choline until 10 m M competitive inhibition was observed; higher concentrations of choline led non-competitive inhibition. The same inhibition was also observed in the presence of TEA or TBA; however, the kinetic model suggested by the authors does not allow for the detection of possible differences between TEA and TBA inhibition patterns. REFERENCES ALDRIOGE W. N. & RE1NER E. (1969) Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate. Biochem. J. 115, 147-162. CLELAND W. W. (1963) The kinetics of enzyme-catalysed reactions with two or more substrates or products. II. Inhibition nomenclature and theory. Biochim. biophys. Acta 67, 173-187. D~SIRI~ B. & BLANCHET G. (1975) Ac6tylcholinest6rase. I. Consid6rations d'ordre cin~tique sur l'interaction avec les effecteurs r6versibles. Biochimie 57, 1347-1357. DC'SIR[~ B., BLANCHET G. & PHILIBERT H. (1973) Activit6 de l'ac6tylcholinest6rase d'erythrocyte/t faible force ionique. Biochimie 55, 643-646. DI~SIRI~B., BLANCHETG., DEFINOD G. & ARNAUD R. (1975) Ac6tylcholinest6rase. II. Aspects exp6rimentaux de I'interaction avec les effecteurs r6versibles en milieu de force ionique 61ev6e. Biochimie 57, 1359-1370. ELLMAN G. L, COURTNEY K. D., ANDRES V. & FEATHERSTONE R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmac. 7, 88-95. HELLENBRAND K. & KRUPKA R. M. (1970) Kinetic studies on the mechanism of insect acetylcholinesterase. Biochemistry 9, 4665-4672. KATO G., TAN E. & YUNG J. (1972) Acetylcholinesterase. Kinetic studies on the mechanism of atropine inhibition. J. biol. Chem. 247, 3186-3189. KRUPKA R. M. (1965) Acetylcholinesterase: structural requirements for blocking deacetylation. Biochemistry 4, 429-435. KRUPKA R. U. 81-.LAIDLER K. J. (1961) Molecular mechanisms for hydrolytic enzyme action. I, II, III and IV. J. Am. chem. Soc., 83, 1445-1460. MOOSER G. & SIGMAN D. S. (1974) Ligand binding properties of acetylcholinesterase determined with fluorescent probes. Biochemistry 13, 2299-2307. MOOSER G., SCHULMAN H. & SIGMAN D. S. (1972) Fluorescent probes of acetylcholinesterase. Biochemistry 11, 1595-1602. PLOWMAN K. M. (1972) En2yme Kinetics. pp. 31-38. McGraw-Hill, New York. PR1NCIPATO G. B., AMBROSlNI M. V., MENGHIm A. R., GIOVANNINI E. & DELL'AGATA M. (1978a) Multiple forms of acetylcholinesterase in ,4 Ilolobophora cali qinosa: Purification and partial characterization. Comp. Biochem. Physiol. 61C, 147 151. PRINCIPATO G. B., GIOVANNINI E., MALLONE L. & MENGmNl A. R. (1978b) Analisi cinetica di un modello di reazione a due siti di legame per l'acetilcolinesterasi. To be published. ROSENBERRY T. L. & BEP,NHARD S. A. (1972) Studies of catalysis by acetylcholinesterase. Synergistic effects of inhibitors during the hydrolysis of acetic acid esters. Biochemistry 11, 4308-4321. STEINBERG G. M. & AMSHEY J. W. (1976) Comparison of the effect of the reversible inhibitor hexamethylenebis-[dimethyl-(3-phthalamidopropyl)ammonium bro-mide] upon the hydrolysis of several substrates by acetylcholinesterase. Archs Biochem. Biophys. 175, 618-626. WILKINSON G. N. (1961) Statistical estimations in enzyme kinetics. Biochem. J. 80, 324-332.