918 CAN. J. BIOCHEM. VOk. 54, 1976

1. Borgmann, U., Moon, T. W. & Laidler, K. J. (1974) 4. Laidler, K. J. (1965) CherniccsE Kinetic-s, 2nd edn, pp. Biochemi.~try I3 ,5 152-5 158 88-90. McGraw Hill Book Co. , New York, N.Y.

2. Borgmann, U., Laidler, K. J. & Moon, T. W. (1975) 5. Laidler, K. J. (1969) Theories of Chenzical Reaction Can. 9. Biochem. 53,1196-1206 Rates, pp. 7679, McGraw-Hill Book Co., New York,

3. Glasstone, S. , Laidler, K. J. & Eyring, H. (1941) The N.Y. T1~em-y qfRabe Processes, pp. 197-199, McGraw-Hill 6. Borgmann, U. & Moon, T. W. (1975) Can. J r Book Co., New York, N.Y. Biochem. 53,998-1004

Binding constants for tetramethylammonium ion determined with irreversible inhibitors of acetylcholinesterase

F. HVERSBN Bllrecrla of'ChemicaE Sqfety, Foods Direcforate, Health Protection Branch, Ott~iwa, Ont., Canada KIA OL2

Received September 26, 1975

Iverson, F. (1976) Binding constants for tetramethylammoniu~m ion determined with irreversi- ble inhibitors of acetylcholinesterase. Can. J . Bio(*klem. 54,918-920

The reversible binding constant (Ki) for tetramethylammonium ion (TMA) was determined from the decrease in the birnolecular rate constant ( k j ) observed with each of 21 organophosphate or carbamate inhibitors of acetyIcholinesterase (EC 3.1.1.7). The Ki values obtained were reasonably constant (5.8 x lW4 + 0.38 M), and this is consistent with reports indicating that TMA binds to a single site on the enzyme.

Iverson, F. (1976) Binding constants for tetramethy%ammonium ion determined with irreversi- ble inhibitors of acetylcholinesterase. Can. .I. Biochem. 54,918-920

Nous avons determink la constante de liaison reversible (Ki) de l'ion tetramCthylammonium (TMA) en evaluant la dininution de la constante de vitesse bimolCculaire ( k t ) avec chacun des 21 inhibiteurs organophosphate ou carbarnate de l'acitylcholinest6rase (EC 3.1.1.7). Les valeurs de Ki obtenues sont raisonnablement constantes (5.8 x 1 0 - 9 0.38 lI4) et ceci confirme les rapports montrant que Ie TMA se lie ii un seul site enazymatique.

[Traduit par le journal]

Introduction TMA binds only to a single anionic site, it is possible

Tetralkylammonium compounds are assumed to bind reversibly to the anionic site of acetylcholines- terase (EC 3.1.6.7), thereby hindering the approach of other active site - directed compounds ( i .e . substrates and inhibitors). Recent evidence (1-3) has shown that there may be more than one anionic site, and Roufogalis and Quist (2) have suggested that TMA binds exclusiveiy to the anionic site, whereas its ethyl homologue (TEA) binds to a peripheral site as well. Another study (3) reported that a ternary complex may be formed with TEA, enzyme, and an irreversible inhibitor. When the re- versible binding constant (Ki) of TEA was deter- mined using the decrease observed in the bimolecu- lar rate constant (ki) of irreversible inhibitors, the Ki values differed by as much as 18-fold. However, if

ABBREVBA'T~ONS: TMA, tetramethylammsnium; TEA, tetraethylammonium.

that the Ki of TMA, determined in the same manner, would not show the diversity observed with TEA. With this approach in mind, the Ki value of TMA was determined in the presence of 21 organophos- phate or carbamate inhibitors of acetylcholines- terase.

hfsrtesiak and Methods

The reaction of acetylchslinesterase (Er with an irrever- sible inhibitor (1) proceeds according to the following scheme:

a k 2

[I 1 E + Pe EP 4 El'

where K, is the binding constant and k , a first-order rate constant governing formation of EI', the inhibited enzyme (4.1- W e n I CK,, the scheme is simplified to

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phos

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S-es

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6.

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1-na

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-met

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5 3,

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trim

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06

is t

he

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ula

r ra

te c

onst

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in t

he

abse

nce

of

TM

A.

bk (a

pp

) is

th

e bi

rnol

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con

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th

e pr

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MA

. C

&

is t

he e

qu

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riu

m co

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r TT

MA

. Th

e m

ean

Kj f

or t

he o

rgan

oph

osp

hat

es is

5.6

x 1

0-4

+ 0.

33 M

; for

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ates

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x 8

W4 f 1

.3 M

. Th

e m

ean

K,

for

all

com

pou

nd

s is

5.8

x

+_

0.3

8 M

.

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928 CAN. 3. BIOCHEM. VOL. 54, 1976

where ki is the bimolecular rate constant. W e n inhibition occurs in the presence of a reversible competitive in- hibitor. the following sequence is added to Scheme 2:

where K , is the reversible binding constant and A the reversible inhibitor. which is TMA.

In the present communication, k , was calculated from the slope of a plot of the log of the remaining enzyme activity vs . time (i.e. log 1% vs, t ) . according to the relation- ship derived from Scheme 2 (9.e. (2.3 log ~ ~ , , / v ) / t ) . where v, is the velocity of the uninhibited enzyme and at is the velocity after inhibition for time t .

In the presence of TMA, k, is determined in the same manner, except that the slope of the plot of log I: 1,s. t now yields an apparent k , , where k i (appi is related to R , through the following expression:

[41 ki(app) = k,K,l(K, + A).

The value of Ki can then be determined, since k,(app), k , , and A are known.

Inhibition of acetylcholinesterase was carried out at pH 7.6. 2 " C , using the reaction vessel described previously (5). Assay of remaining enzyme activity was accomplished with a pH Stat and 3 mM acetylcholine chloride as sub- strate.

The inhibition procedure was repeated with 1 mM ThlA present during the incubation of enzyme and inhibitor. This concentration of TMA was selected since preliminary work suggested that it would probably exceed Mi and pro- duce an acceptable decrease in the apparent K i value re- quired to evaluate Ki. Unlike TEA and other quaternary compounds, TMA has been shown to have Battle effect on acceleration of inhibition or decarbamylation (6,7). a pre- requisite for the present study. Acetycholinesterase weas obtained from Sigma Chemical Co., St. Louis, M 8 .

Results and Discussion Table 1 lists the irreversible inhibitors used and

the values found for the inhibition constants. Al- though the size of the leaving group varies consider- ably, the K , values obtained for TMA appear reasonably constant and there does not appear to be a consistent difference between organophosphate or carbarnate inhibitors. The mean Ki for all com- pounds is 5.8 x 10-+ 0.38 M .

The greatest deviation for Ki was obtained within

the carbarnate series where carbaryl yielded a K , value threefold higher than the overall mean. The low water solaibility of this compound precl~ided further work in establishing the effects of TMA on the individual affinity and carbamylatlon rate con- stants (i.e. K , and k,, Scheme 1 ) . It was hoped that 4-hydroxyl carbaryl, which contains the added hy- drophilic moiety, would allow determination of' the additional constants, beat again solubility was too Iow. However, the K , value determined for the 4-hydroxy analogue was twofoId higher than the mean Ki.

The present study is based on a simplified reaction sequence which avoids effects of TMA on individual reaction constants. On the other hand, the sequence predicts that K , values for TMA should be constant unless a ternary complex can be formed. Since the Ki values were found to be quite similar, the present results are consistent with the proposal that TMA binds 80 a single site.

It is interesting to note that the compounds elicit- ing the greatest deviation in Ki contain the naphthol leaving group. Wosenberry and Bernhard (8) have shown that certain naphthol derivatives are unique in that they may form a ternary complex with the enzyme and substrate. It was also reported that I-naphthyl acetate acted synergistically. The larger K , values observed for carbaryl and its analogue suggest that TMA is Iess effective in preventing inhibition by these compounds, brat any furthrar in- terpretation requires a more extensive kinetic analysis.

1. Kitz, W. 3 . . Braswell. L. M. & Ginsberg, S. (1970) ~%f4d . Pharmacol. 6 , 108-121

2. Roufogalis, B. D. & Quist, E. E. (1941) ~%foi. Phar- mat-ol. 8,41-49

3. Iverson. F. (1941) Mok. Pharrnacol. 7 , 129-135 4. Main, A. R. (1964) Science 144,992 5. Iverson, F. & Main, A. R. (1969) Biochemistry 8 ,

1889- 1895 6. Metzger, H. P. & Wilsont I. B. (1943) J. Biol. Chem.

238,3432-3435 7. Roufc~galis, B. D. & Thomas, J . (1970) J. Pharrn.

Plzcarmacol. 22,449-658 8. Rosenberry, T. L. & Bernhard, S. A. (1971)

Bic)chc.mistr-y % I , 43084321

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