The reactivation of carbamate-inhibited cholinesterase, kinetic parameters

PESTICIDY

BIOCHEMISTRY

AND

PHYSIOLO(‘rY

The

Reactivation Cholinesterase,

W. Department

3, 1%&130

DOUGLAS

of Carbamate-Inhibited Kinetic Parameters REED’

AND

T. R.

FUKUTO

of Entomology, Division of Toxicology and Physiology, University of California, Riverside, Calijomia 92502

Received

November

13, 1972;

accepted

January

29, 1973

The rates of spontaneous regeneration or decarbamylation of fly-head and bovine erythrocyte cholinesterase inhibited by methyland dimethylcarbamic acid esters were determined under different condit,ions of pH, salt concentrations, and temperature using Sephadex gel filtration as a means of isolating the carbamylated enzyme. The pa-rate profiles for decarbamylation of the two choline&erases for both methyland dimethylcarbamylated enzyme had maximum rates at pH 8.5-8.9 for bovine erythrocyte and pH 8.1-8.5 for fly-head cholinesterase. Imidaxole, hydroxylamine, and inorganic salts did not alter the decarbamylation rate. Values for the energy of activation for the decarbamylation reaction indicate a somewhat unstable and more react.ive carbamylated enzyme for bovine cholinesterase compared t.o fly-head cholinesterase, but entropy factors are more favorable for decarbamylation in the latter. Regeneration rates in deuterium oxide for both enzymes and bot.h methyland dimethylcarbamatea were approximately six to seven t,imes slower than in water, indicating a secondary isotope effect. A mechanism for decarbamylation consist,ent with the dat,a is cited,

where IX is the carbamate ester, E is free AChE, X is a leaving group, EIX is the reversible complex, EI is carbamylated enzyme, and P is alkylcarbamic acid. During the assay of AChE activity itl uifro the primary form of inhibited enzyme is most probably t,he carbamylated cnzymc, although the reversible complex Gertainly exists (6). Tt is the decomposikm of the carbamylated enzyme which is responsible for the eventual return of enzymatic activity after the effect of the residual carbamate has been minimized or removed by dilution or gel filtrat,ion (1, 4, 7).

INTRODUCTION

The inhibit,ion of acetylcholinesterase (AChE) by esters of alkylcarbamic acid has been shown to result from the ability of the carbamate t’o act as a poor substrate (l-6). Equation 1 depicts the overall mechanism of the inhibition process and it appears that inhibitory action is attributable mainly to t,he slow turnover rate which is determined by the jza step, E+IXwEIX-

h

k,

k-1 EI A

x k,

E + I’,

(1)

1 Present address: Department of Physioiogical Chemistry, The Johns Hopkins University School of Medicine, Raltimore, Md. 21205. This investigation was supported by NIH Training Grant ES 47

from the National Health Sciences and from the Environmental ington, DC. 120

Copyright All rights

@ 1973 by Academic Press, Inc. of reproduction in eny form reserved.

Institute of Environmental by Research Grant R801837 Protection Agency, Wash-

REACTIVATION

OF

CARBAMATE-INHIBITED

The suggestion has been made by Bender and Kezdy (S), among others, that acylation and deacylation of chymotrypsin proceed via similar mechanisms. The two analogous steps in the carbamylation scheme, steps k? and ka, may also proceed similarly. In view of this, a comparison of decarbamylation to the dcacetylation step in ACh hydrolysis should he of mrchanistic inter&. Previous studies on the spontaneous rcactivat’ion of AChE inhibited by carbamat’e &em were carried out by means of dilut,ion techniques and in the absence of the subst8rate acetylcholine (or acetylthiocholine) (1, 4, 5j. In the prcscnt study, the carbamylatcd AChE was separated from cxccss inhibitor by Scphadex gel filtration and decarbamylat8ion of the inhibited enzyme was determined in the presence of t#he substrate acrtylthiorholinc.

CHOLINESTERASE

121

carbamate (2), bp 12s122°C (0.2 mm), was prepared according t’o Fukuto et al. (12). The synthesis of ring-labeled trit.iatrd carbofuran (4,5,6-H3-2,3-dihydro-‘,8dimethylbenzofuranyl-7 mrthylcarbamatr), mp 149-l:i2°C, has been described elscwhere (13). Carbonyl-labeled 14C Bawl (6-chloro-3,4-xylyl methylcarbamate) was supplied by the Upjohn Co., Kalamazoo, 111. Isotope eflect. The solid components of the assay system, AChE, DTNB, and ATCh, were dissolved in 99.SS% D20 (Inbcrnat.ional Chemical and Suclrar Corp.). The buffer for the system was prcpared by dissolving anhydrous sodium carbonate in D20. This gave a pD (analogous to pH) nrar 11.0. Thr pD was lowered t#o 8.42 by passing dry carbon dioxidr t,hrough the solution. The pD of the result,ing deutcro sodium bicarbonatr was determined by use of the pH correction JIATERIALY AND METHODS formula of E’ife and Bruice (14), and a Bovine eryt’hrocyte acetylcholinesterase Beckman Expandomatic pH mrtrr. r\Tmr (sp act 3.5 units/mg) was purchased from analysis for water showed that the buffrr Sigma Chemical Co., St. Louis, Mo. The solution was 99X% deutwium oxide. housefly-head AChE was prepared from Kilretic pwcedure. After the residual heads of Musca domestica Linn. harvested carhamatc was removed from the AChE by the method of ;\Ioorefield (9). The heads by Scphadcx gel filtration, an aliyuot was Lverc then homogenized in 0.1 N potassium added t,o the reaction ruvct,tc and hgchloride in a glass tissue grinder. This drolysis of t.hc substrak was monitored homogcnatc was centrifuged at 15,000 !/ for according to t.hc procedure of Ellman et al. (lo), at 412 1l1l.c.Thc~ l<:j step [Eq. (l)] 60 min in a Sorvall Superspcc~dRC-2 centrifugc held at, 4°C’. The supcrnat,ant fluid indicatw that dccarhamylation will result from this spin was used as the cnzgmc in a first-order incrcasc in AChE (or R decrease in carbamylatcd cnzymcj. Since source. The AChE assay employed \va,s that of the amount, of AChE present in succcssivc Ellman et al. (10). The substrate, acctpl- time intrrvals is linearly related to the change in absorbance at, 412 nip, it follows thiocholinc iodide (ATChj, was synthcsized according to Rmshaw et al. (II), and that as AChE conccnt,ration increases substrate hydrolysis should accelcratc and the the rcagcnt, 5,5’-dithiobis-2nitrobenzoic acid (DTKB), was purchased from Ald- plot of absorbance vwsus time should have rich Chemical Co. a positive curvature. The plot would have Carbaunate inhibitors. The carbamates the form of an inkgrated first-order cquaused, t,ogether with their sources, are in- tion, i.e., dicated below. 3-Isopropylphenyl methyly = At - A/k(l - eek’), (2) carbamate (I), mp 7577”C, was supplied where y is absorbance at time t, A is the by the Hercules Powder Co., Wilmington, rate when all the enzyme is in the uninDE. 3-Isopropyl-4-nitrophcnyl dimethyl-

122

REISDANDFUKUTO

hibited form, and k is equal to the constant of acceleration or the regeneration rate constant. The value of A, the limiting velocit’y for ATCh hydrolysis, was wtimated from the absorbance versus time plot after the curvilinear lint became linear. In all cxperiments t’hc regeneration wartion was followed until this relationship was linear com\vit’h time, i.c., \vht:n no accclcration poncnt, in t,hc curve was detwtablc. The data also may bc tittcd to the following multicompartmcntal mathematical model and this model was employed in this study to obt#ain t’hr decarbamylat)ion constant (k7), which is idcnt,ical to I; in Eq. (2j. I;,,’ 0) L

-

k, (C) __+

(E:) k,’ 1 (11’)

(3)

(B) - kr In this model, compartmtntJ (A) is inhibited enzyme, (B) is frw cnzymc, (C) and (IX,) arc the couplrd substrate reactions, (D) is free enzyme at t = 0, and (I’) is optical density. The appropriate rate constant’ for rcgrncration of free cnzymc is k,, while the /c, and k,’ are indicat,ivc of the reaction of enzyme with the substrate (ATCh) and the k, and k,’ arc charactrristic of the rrartion of this substrate hydrolysis product and the rragcnt DTSB. The data fitting to the model [Eq. (3)] was donr ut.ilieing the syst,cms analysis and modeling program of Berman and Weiss (15, 16). In this least-squaws program, upper and lower limits for k, wcrc set by rmploying values selcctcd from previous stud&, c.g., Rcincr and Aldridgc (5). The values so chosen wcrc 0.001 rninr and 0.300 mm’ for the louver and upper limits, wspectivcly, for both carbamatc dcrivativcs of AChE. This analysis \vas most kindly performed on a Univac 1108 comput,rr by Dr. nlones Berman, Kational Cancer Institute, Thcorctical Biology Laboratory, National Institutes of Health, Bethesda, ,IId. The gcncral procedure for measuring dccarbamylation rates is as follows: A l-ml (4

aqueous solution of enzyme was placed in a 3-ml vial. To t’his was added 20 ~1 of an acetone solution of the inhibitor. Thr cnzymc concentration in terms of spwitic activity varied bctn-ecn the fly cnzymc and the bovinr enzyme, but the concentration of all inhibitors remained at 2 X lop4 JI. Each of the cnzymc preparations used was complctcly inhibited when they wrre subjcctcd to this c,oJlct,lltrat.iol1. The v~~zyrnvinhibitor solution was incubutcd at rooni tcmpcraturc: (Z”C) for 15 min. The solution was then applied to t’hc surface of a Scphadex G-25 or G-10 column and clutcd wit,h water. This column was I.7 cm in diameter and packed to approximatrly 11 .O cm in height. The positions of the enzgmc and inhibitor during the passage n-cw monitored by Dcxtran blue (Sephadex) :md p-nitrophcnol, resp&ivcly. The clutcd (‘IIzymc was collrct,rd in a small (3-ml) vial and an aliyuot of 100 ~1 \vas withdrawn with two fillings of a SO-~1 sgringc (Hamilton co., Whittier, (:A’). This aliquot I\-as in jcctcd into a cuvettc containing :‘.s ml Tris buffer, pH 7.9, 0.2 ml DTlYB (0.01 M), and 40 ~1 0.75 ;lI solution of XTCh (0.01 Jr). The cuvcttc was stoppcwcl, inverted, and placed in the constant ttmperaturc ccl1 holdrr of a Unicam Sl’ SO0 ult,raviolt+ spc~ctrophot,orn~~t,c,r. The cuvcttcb holder \\-a~ hrld at, constant trmpcrat~urc by circulating watrr from a Haakc Mod(~l 1”~ thwmostatic bath. A solutJion containing all rcagents cxcrpt for the enzyme was plawd in the refcrcncc cell. The absorbance at 41’1’ mp was then rccordrd at l- or &mm intcrvals until the cxperinicnt xvas twminatcd. HEC;ULTS ,

+

Since this is a study of the regeneration reaction [ka, Eq. (1) or k,, Eq. (3)], it was of fundamental importance to be sure that, the carbamylated enzyme was bring isolatcd from noncovalcnt,ly bound carban1at.c. The crucial point, therefore, was to d&rmine the proper functioning of the Scphadex column. The first control cxperimcnt was to asccrt,ain the volume that would

REACTIVATION

OF

CARBAMATE-INHIBITED

CHOLINESTERASE

123

precede the protein fraction \vhen a 1.0-1~~1 sample was applied. The position of the protein was readily determined by the addition of the dye, Dextran blue, which is clxcluded from all Scphadex gels and, t,herefore, flows with the protein. The wzymatic act)ivity of this fraction n-as assayed by withdrawing an aliyuot and adding it) to a roac%ion mixtuw. Oncl-millilit~t samplw \vore collwtt~d starting at the first :tppo”rancc

(Jf

blue

(!dc11.,

the

dor

con-

tinuing for four successive milliliters of cgluant. The majorit,y of the activity occwwd in the first fen millilitrrs aft,er appraranw of t,hcAcolor. This experiment indicatw two important points. First, the passage through the column apparently dew not alt’cr the activity of thr enzyme and second, the enzymatic activity is associatod with the Dextran blue fraction, as was cxpect,cd. The scheme in Ey. (1) indicates t,hat the Iclaving group (X) should be lost from the enzyme during the inhibition process. This was checked by using carbonyl-labeled 14CHanol and ring-labeled 4,5,6-H3-carbofuran. When Bawl was used as an inhibitor, radioact,ivity was associated with the VIIzynw frac:t,ion ; howrvcr, when carbofuran was used, no radioact’ivity appeared in thcb canzymc fraction. O’Brien (3) observed the samcx type of c&r ,cleavage when he inhibit,cd bovincl AChE with “H-3,5-diisopropylphenyl methylcarbamate. In this case, a “burst” of radioactive diisopropylphenol was libcratrd when AChE and carbamatc, wwc mixed. Thp ‘%urst” was followed by- an appcaranw of radioactivit! which was linear with tinw. The cvidtww citcld abow providw adequate basis for the assumption that the carbamylated enzyme was being isolated. The increase in enzyme activity with timt, reprcaents dwarbamylat.ion. This dccarbamylation reaction was considcwd from th(x following points of viwv. pH-Rate dependence. Since different mechanisms appear to be operating in the base-catalyzed hydrolysis of methyl- and

Flc;. J. Etiwl of pH noninhibikd howfly-head

on subkalo hyt1rolysl.s acutylcholin~stcras~.

by

dimethylcarba,mattas (3, 12, 17), it, was wnsidered worthwhile to determine the effect of pH on the regenerat’ion reaction of methyland dimethylcarbamyl AChE. The accepted role of carbamates as altflrnate substrates for AChE predicts that the decarbamylation reaction might have the same pH-rate profile as noninhibited substrate hydrolysis. The substrate hcrc, acetylthiocholine (ATCh), has been shoun (18) to have a pH-rate profile which gives a plateau at high pH in contrast to the bellshape curve found in ACh hydrolysis. The platrau was not obstrwd in this study (Figs. 1 and 2 for ATCh hydrolysis b> bovine eryt,hrocyte or fly-head AChE). Th cbxplanation given for the plateau previously observed with ATCh is based on the weak hydrogen-bonding property of th(b sulfu;. atom and, therefore, loss of the proton in the acidic group in the esteratic site at

A

FIG. 2. Effect inhibited bovine

R

of pH on substrate hydrolysis rrythrocyte acetylcholinesterase.

by non-

124

REED

AND

FUKUTO

I

I

/

7.0

8.0

90

I

10.0

PH

FIG. 3. Effect by 3-isopropylphenyl

of pH on the decarbamylation rate constant methylcarbamate.

high pH should not significantly decrease the rate of ATCh hydrolysis. However, the acetyl-enzyme is the same whether ATCh or ACh is used as substrate, and therefore, pH dependence should be the same if the Ic, step is rate limiting; although this may not be the case with fly-head AChE (R. M. Krupka, personal communication). The pH-rate profiles for decarbamylation of a methyl- and dimethyIcarbamyi bovine erythrocyte AChE inhibited by 3-isopropylphenyl methylcarbamate (1) and 3-isopropyl-4-nitrophenyl dimethylcarbamate (2) appear in Figs. 3 and 4, respectively. The maximum rate is found between pH

I

I

I

70

80

90

I

10.0

PH

FIG. 4. Effect of pH on the decarbamylation rate of bovine erythrocyte acetylcholinesterase

constant inhibited bamate.

by

,$-nitro-S-isopropylphenyl

dimethylcar-

of

bovine erythrocyte

acetylcholinesterase

inhibited

S.6 and 8.9 for both of the figures. The corresponding regeneration of methyl- and dimethylcarbamyl derivatives of fly-head AChE appear in Figs. 5 and 6, respectively. In this case the methylcarbamyl derivat’ive has a maxima between pH 8.0 and S.5 and the dimethylcarbamyl enzyme at pH S.3-S.S. It may be noted that the regeneration rate constant for the methylcarbamyl AChE has a more precipitous decline with increasing pH for both the bovine and housefly enzyme than do the dimethylcarbamyl AChE derivatives. Activation parameters. Data showing the effect of temperature on the rate constant, for regrneration of housefly and bovine erythrocyte AChE inhibited by 3-isopropylphcnyl methylcarbamate (1) and 3-isopropyl-4-nitrophenyl dimethylcarbamatc (2) are presented in Table 1. Included in the table are the values for the Arrhenius activation energy (E,) calculated by rcgression analysis of a plot of log Iz, vs l/Z (“I<), and values for the entropy of activation (AS*). Satisfactory Arrhenius plots were obtained with correlation coefficients ranging from 0.94 to 0.99. Values for the regeneration constants (k7) show that the rate of regeneration of housefly AChE inhibited by 1 or 2 is about twice as fast as the corresponding bovine erythrocyte enzyme (compare rate con-

REACTIVATION

Rate Constants

(k,) and Activation phenyl Methylcarhamate

Enzyme

OF CARBAMATE-INHIBITED

Paramctcrs for th(j Regeneration and Y-Isopropyl-4-nitrophwqyl

Carbamate 1 3-Isopropylphenyl methylcarbamate

22.0 26.0 29.5 32.5

Fly

2 3-Isopropyl-4nitrophenyl dimethylcarbamate

18.5

Bovine

1 3-Isopropylphemyl methylcarbamate

Bovine

2 3-Isopropyl-4nit,rophenyl dimethylcarbamat’e

--___.

of Znhihitpd Cholineslerase Dinwthylcarbamate at pH k,

TP.2

Fly

Ea

by 3-Isopropyl7.9

(kcal/mole)

AS*

(e.u.)

25.7

11.3

27.9

17.2

0.105

26.0 29.5 32.5 18.5

22.0 26.0 33.0 18.5

(min-1)

0.0258 0.0465 0.104

22.0

26.0 33.0

125

CHOLINESTERASB

0.00735 0.0135 0.0225 0.0400 0.0707 0.00539,0.00583 0.00675,0.00863 0.0200 0.0242 0.00394,0.00450 0.0122 0.0142

19.8

-2.8

16.4

-3.5

-- -

at, 26.O”C). The values of k, (min-‘) for the spontaneous recovery of the methylcarbamyl and dimethylcarbamyl bovine AChE at, 2VC arc 0.020 and 0.012, respectiwly, in good agreement with t’hu values of 0.0234 and 0.0123 (25°C) rcported by Reiner and Aldridge (5) for the same enzyme, alt,hough determined by a diff crcnt’ procedure. The magnitude of E’, and AS& for the rcgcncration of housefly and bovine AChE reveal distinct differences between the two enzymes. E’, for the regeneration of carba&ants

0011

7.0

mylated housefly AChE is substant’ially greater t,han that for bovine AChE, in spite of the faster rate of decarbamylation by the former. Evidently, the larger act,ivation energy for housefly AChE is compensated by favorable entropy effects. The activation energies for decarbamylation of housefly and bovine AChE are larger than the 14 kcal/mole observed for the alkaline hydrolysis of p-nitrophenyl methylcarbamate (12), but they approach those reported for the alkaline hydrolysis of ethyl methylcarbamate and dimethylcarbamate (17). This is consistent with the gen-

I

8.0

70

FIG. 5. Effect of pH on the decarbamylation rate constant for housefly-head acetylcholinesterase inhibited by 3-isopropylphenyl methylcarbamate.

80

90

100

PH

PH

FIG. 6. Effect of pH constant for housefly-head by .&nitro-S-isopropylphenyl

on the decarhamylation rate acetylcholinesterase inhibited dimethylcarhamate.

126

REED

Effect

of Salts

on Regeneration

Rates

--__ %Isopropylphenyl methylcarbamate

3-Isopropyl-4nitrophenyl dimethylcarbamate

FUKUTO

TABLE

3

of Carbamate-Inhibited

Inhihit.or __-

AND

Bovine

Media

0.1

Acctylcholinesterasc

k,. (min I) -

0.05 Tris pH 0.05 Tris pH 0.1 :V LiCl 0.05 Tris pH 0. I N NaCl 0.05 Tris pH 0.1 N KC1 0.05 Tris pH 0.05 Tris pH 0.1 LiCl 0.05 Tris pH

Erythrocytc

~~-__

Kelative rate

7.9 7.9

0.018’2

f

0.0006

o.os2

f

0.0041

7.9

0.033

f 0.0017

1.8

7.9

0.032

f 0.0041

1.8

7.9 7.9

O.(Y20 zfz0.0010 0.029 f 0.0010

1.4

7.9

0.0’29 f 0.0010

1.4

0.0:<4

1.7

1.0 1.8

1.0

NaCl

0.O.i Tris pH 7.9 0.1 KC1

cral belief that carbamylation, acylation, and phosphorylation takes place on an aliphatic hydroxyl moiety in t’he active site, i.e., the serine hydroxyl. Salt e$ects. The primary and secondary salt effects on reaction rates have been well investigated (19, 20). Mow recently Bruicc et al. (al), have investigat.cd the structure-forming properties of different salts on aqueous transition states. While the interpretation of primary or secondary salt, eff ect,s could lend information regarding the mechanism of decarbamylation, pronounrrd salt effects were not found in this study. Thrre was probably a sncondary nonspecific salt c>ffcct which is consistent with acid-base catalysis, but this is to ho cxpectrd. Table :! lists the salt clffect on the regencrat,ion constants for bovine cnzyme that had been inhibited by 3-isopropylphenyl methylcarbamat,e and 3-isopropyl-4-nitrophenyl dimct’hylcarbamatc. Deuteriwn osicle ejfect. Deut,wium oxide has been employed previously in t,hcbdifferentiation of general base and nucleophilic catalysis of hydrolysis (‘L--24). The values of kH/kD arr generally 1~s than 2.0 for nucleophilic catalysis due to the secondary isotope effect. Dwterium oxide was used as the sole solvent in the cxpwimc~nts

f

0.001n

cited in Table 3. lnhibitors 3-isopropylphcnyl methylcarbamate (1) and 3-isopropyl-4-nitrophenyl dimethylcarbamatc (2) w,ere used to carbamylate bovincl crythrocytc AChE. The isotope effect of 5.7 for dimethylcarbamyl enzyme regeneration and 7.5 for monomethylcarbamyl enzyme is consistent wit,h a single prot.on transfer mechanism bring operative in both cases and that gcncral acid-base assist’rd nuclcophilic attack by water is probabl:, involved (2.5). Tha magnitude and dircction of the isotope cffrct supports this pwliminary conclusion. While the cffwt of dwtcrium oxide* on waction rate constants has been generally confined to model systems, it was felt worth trying here bccausc the decarbamylat’ion reaction has been shown to be an cbasily measured cstcr hydrolysis. The assessment, of t.he decarbamylation constant (or conversely the acceleration constant for ATCh hydrolysis) does not depend on the intricacies of the hydrolytic nwchanism but, rather on a single-step roaction. It is felt, t’herefore, that t,he deuterium oxide effect might, upon comparison with the mod(tl systems, yicxld information as to the t,ypc: of rcactir~n involved.

REACTIVATION

OF

CARBAMATE-INHIBITED

TABLE Efcct

of Dwterium

127

CHOLINESTERASE

3

Oxide on the Regeneration Rate of Carbamatc-Inhibited Erythrocytc Acetylcholinesterase at pD of 8.42

Bovine

~.,

Inhibitor 3-Isopropylphenyl methylcarbanmte 3-Isopropyl-4nitrwhenvl dimethylrarbamnte _

”

Solvent -__~ - .~___ Water Deuterium oxide Water T)euterium oxide

DIscussloY I

The method used in this study of the regeneration of AChE inhibited by methyland dimethylcarbamate esters differs from t.hat] normally used (1, 5) in two basic rcspccts. First, excessinhibitor (carbamatc) was separated from the enzyme-carbamyl enzyme mixture by Scphadex chromatography. The general procedure used to minimize or t,erminatc continuous carbamylation has been t.he “dilut,ion” t,echniyue. Second, the rate of spontaneous regeneration of the carbamyl enzyme was monitored in the presence of the substrat’e ATCh by following the increase in the velocity of substrate hydrolysis with time. As expected, a posit’ive curvature was obt#ained in t,he relationship between mzyma& activity and time. In the method normally used, the diluted cnzymc-inhibitor mixture is allowed to stand and aliyuot samples are taken for assay of enzymat’ic activity after fixed time intrrvals by addition of substrate (ACh or ATCh). This method rcyuires that initial velocities for substrate hydrolysis be used to c,alculat,e the first-order regeneration constant since the velocity surely will increase with time as rcgencrat’ion occurs. The major difficulty in the present method resides in the proper analysis of the data. For this purpose a mathematical model was established and regeneration const.ants (k,) were calculated by computer analysis. Another problem lies in the possibility that the reagents used to estimate subst,ratc: hydrolysis, i.e., ATClh, DTNB

k,,/ku

kr 0.0.50X 0.00674 0.0345 0.0060

f f f f

0.0048 0.00033 0.00070 0.00046

7.5 .i.7

and its products, may have some effect’ on the regeneration rate. This may be especially true with respect. to the substrate (ATCh) since it has been shown (26) that decarbamylation of methylcarbamylated AChE is inhibited by acetylcholine. However, in spite of these possible effects, the values obtained for k, by this method are similar to those reported by other invcstigators (1, 5, 27-29). Llodels for the hydrolysis of ACh by AChE propose that bot,h acetylakm and deacetylation of AChE arc mediat’ed by acid-base catalysis (30, 31). The mrchanism of dcacetylation is suggested as being the reverse of acetylation. The result’s from t.his study using 14C-labeledcarbamates and of t’he effect of pH, deutcrium oxide, and salts on the regeneration of carbamylated AChE suggest that a similar mechanism is in operation in the carbamylation and dorarbamylation of AChE by carbamatc esters. A mechanism for t#hese procc~sscs consistent with that proposed for ACh hydrolysis is present)cd by the scheme in Fig. 7. Although t.hc artual inhibition rcaction was not examined in t#hisstudy, the scheme includes the carbamylation process based on analogy wit#h the model for ACh hydrolysis. In particular, the effect of pH and deuterium oxide indicates that dccarbamylation is acid-base catalyzed as depicted in the scheme. The pH-rate profiles for decarbamylation for mct.hyl- and dimethylcarbamyl AChE is compatible with a mechanism in nhich a water rrrol~cule attac*ks the carbony1 carbon of the carhamate moiety with

128

IWED

AND

E+I

FUKUTO

EI

EI'

!EI'! FIG. 7. Model for the reaction of carbamic acid EI = reversible complex, EI’ = CarbamyGAChE, mic acid (unstable).

esters with acetylcholinesterase. (EZ’) = transient intermediate,

assistance by the basic (imidazolc) and acid (tyrosine OH) groups in t)he active site. This sort of bifunctional mechanism would not develop a formal charge and so a salt effect would be expected to be slight, if exist’ent. The relatively small salt effect (cf Table 2) is perhaps consistent in this regard. The dcuterium oxide effect of 5.7-7.5 for kH/lcD is of the magnitude cited for a kinetic isotope effect which would be consistent with a proton transfer being rate limiting. The values of 6-10 for kff/k~ in the reactions of normal proton transfer with nucleophiles has been cited as indicative of nucleophilic attack involving proton transfer (22, 23, 32-34). The dcuterium oxide effect observed in this study is consistent with water being directly involved in decarbamylation as indicated in the scheme. Assuming that a mechanism for decarbamylation such as t#hat given in Fig. 7 applies, one might not expect to note a large change in Ic, when different nucleophiles are added because the decarbamylat#ion is catalyzed internally by groups within the active site. It should be nobed with reference to the data in Table 1 that hydroxylamine and imidazole at 10m4 M did not change the rate constants for decarbamylation for either enzyme. These results in-

E = AChE, I = carbamate, I’ = N-substituted carba-

dicat’e that at this concentration t’he added nucleophiles are not able to orient at the active site as are the catalytic groups in the enzyme. Wilson et al. (27) demonstrated that 1 M hydroxylamine added to the carbamyl enzyme increased decarbamylat,ion about 2-fold. It, is possible that the concentration of added nucleophile in our study was not saturating with respect to the carbamyl enzyme. However, at, concentrations as high as 1 M hydroxylamine, it appears that the nucleophile is directly att’acking the carbonyl carbon instead of assisting the water attack. While this would give an effect, it would be specific t’o the added nucleophile and not the spontaneous reactivation and, therefore, was not pursued further in the present work. The results of the pH-rate studies eliminate OHcatalysis and thus water remains the most likely nucleophile. The values for the activation parameters (Table 1) indicate that housefly-head and bovine erythrocyte AChE are definitely different’, in spite of the fact that, comparable k, values were obtained with each. The large activation energy and more positive entropy of activation observed with housefly-head AChE suggests that there is greater conformational assistance in decarbamylation in this case.

REACTIVATION

OF

CARBAMATE-INHIBITED

REFERENCES

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