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FROMDistribution authorized to U.S. Gov't.agencies and their contractors;Administrative/Operational Use; JAN 1985.Other requests shall be referred toCommander, US Army Medical Research andDevelopment Command, Attn: SGRD-RMS, FortDetrick, Frederick, MD 21701-5012.

AUTHORITY

Fort Detrick memo, 6 Aug 1996

THIS PAGE IS UNCLASSIFIED

[OTC FILE C(WY C* AD

Acetylcholinesterase and Acetylcholine Receptor

N--4% Annual Reoort

N DTIClo Saul G. Cohen, Ph.D. ELECTE

January 21, 19850 D

Supported by

U.S. ARMY MEDICAL PESEARCH AND DEVELOPMENT COMMANDFort Detrick, Frederick, Maryland 21701-5012

Contract No. DAMD17-83-C-3251

Brandeis UniversityDepartment of Chemistry

Waltham, Massachusetts 02254

Distribution limited to U.S. Government agenciesand their contractors; Administrative/Operational

Use, May 21, 1986. Other requests for this documentshall be referred to Commander, U.S. Army MedicalResearch and Development Command, ATTN: SGRD-R4S,

Fort Detrick, Frederick, Maryland 21701-5012

The findings in this report are not to be construed"as an official Department of the Army position

unless so designated by other authorized documents.

.: ~ ~87 0•

SECURITY CLA9S5FICATION OF rH14S PAGE

REPORT DOCUMENTATION PAGE 0711' OM8 NO. 070"1088Is. REPORT SECURITY CLASSIFICATION 1b, RESTRICTVE MARKINGS

Unclassified________________________2a. SECURITY CLASSIFICATION AUTHORITY 3D.ISTR1.&U'TION/'AVtI.LA~tLeTY OFRR

ilstri ution limte to Gvrenaen2b. oIDcLSSIFICATIoN I OWNGRADING SCHEULE and their contractors; Administrative/

Operational Use, May 21, 1986.

4. PERFORMING ORGANIZATION REPORT NUMBER(%) 5. MONITORING ORGANIZATION REPORT NUMBER(S)

66. NAME OF PERFORMING ORGANIZATION 6b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATIONBrandeis University I (~Department of Chemistry_______________________

6C. ADDRESS (CRtY, State, and ZIP Code) 7b. ADDRESS (City, State, and ZIP Code)

Waltham, Massachusetts 02254

So. NAME OF FUNDING /SPONSORING 8b. OFFICE SYMBOL 9. PROCUREMENT INSTRUMENT IOENTIFICATION NUMBERORGANIZATION U. S. Army Medica (if apkb) DAMDl7-83-C-3251

Research & Development Command&L. ADDRESS (01ty, State, and ZIP Cod) 10. SOURCE OF FUNDING NUMBERS

Fort Detrick PROGRAM PROJECT ITASK WRK UNITFrdrik arlnd271-02ELEMENT NO. NO. 3M-1 [ NO. CCESSION NO.!Freerck Mryan 270-51261102A 61102BSI1 EF 001

11. TITLE (include Securfty Ouuuifiation)

Acetylcholinesterase and Acetylcholine Receptor1.PERSONAL AUTHOR(S)

Saul G. Cohen, Ph.D.

13a. TYPE OF REPORT 113b. TIME COVERED 114. DATE OF RIEPORT (Year,MonthM, Day) IS.- PAGE COUNTAnnual Report FROM 83/9/15 TO 84/9/14 1985 January 21 I 27

16. SUPPLEMENTARY NOTATION

17. COSATI CODES 18I. SUBJECT TERMS (Continue an revier if neconuay and identify by block finumber)FIELD IGROUP ISUB-GROUP

_nfi Acetyicholinesterase, Active Site,Label,Inhibitors

19, ABSTRACT (Con tinue an reerse if necessary and identuf' by block number)

We seek information about the properties and amino acidcomposition of the active site of acetyicholinesterase (AcChE).Work is based on the view that the part at which the positivelycharged fl-trimethylammonio substituent of acetylcholine (AcCh)binds is not anionic but is uncharged and complementary to thetrimethyl character of that substituent. The subsite would bemore specifically explored by uncharged reagents, while posi-tive reagents would react with peripheral anionic groups.AcChE was isolated from Torpedo nobiliana by affinity chromato-graphy, gel electrophoresis, and autoradiography after 3 H-DFPlabeling showed satisfactory purity and the major band at -70lcD. Sigma eel AcChE showed much extraneous protein, and was

20. DISTRIBUTION /AVAILABILITY OF ABSTRACT 21 ABSTRACT SECURITY ':LASSIFICATIONC3UNCLASSIFiEDfU NLIMI TED (0 SAME AS RPT C3 DTIC USERS Unclassified

22s. NAME OF RESPONSIBLE INDIVIDUAL 22b TELEPHONE (incude Area Codeti 22c. OFFICE SYMBOL01le 66ý 3- 7 325 tSGRD-RMI-S

>~00 Form 1473, JUN 86 Prev.ious edtitons are obsolete. SECURITY CLASSIFICATION OF THIS PAGE

19. ABSTRACT(cont.)

suitable for kinetic studies but not for study of amino acidcontent. Initial emphasis is on labeling with 1-11 C-l-bromo-pinacolone, (CH3 ) 3 CCO14CH2 Br, which will be prepared in 60pmole quantity. Other potential irreversible labeling agentswere studied briefly: chloromethyl pivalate, (CH3 ) 3 CCO2 CH2 Cl,chloromethyl acetate, CH3 COZ CH2 Cl, methyl trimethylammonio-acetate, (CH 3 ) 3 N* CH2 CO2 CH3 , tert-butyl peracetate, (CH3) 3 C-OOCOCH3 , methyl methanesulfonate, CH3 SO2 0CH3 , methyl benzene-sulfonate, C6 H5 SO2,CH 3 , and styrene epoxide, C6 HOCý;H 2 . The

methylsulfonyl and trichloromethyl groups were examined inreversible inhibitors, methylsulfonylethyl acetate, CH3 S(O2 )-CH2 CHM OCOCH3 , trichloroethanol, Cl 3 CCOH, and chloral hydrate,C13 CCHO.H 2 0. Nitrobenzene was an effective reversible inhibi-tor and polar interactions at the aryl binding site will bestudied.

A study of hydrolysis of fl-substituted ethyl acetates, f-X-CH2 CH2 0COCH3 , by AcChE was published. Reactivity, normalizedfor effect of the fi-X-substituent on intrinsic hydrolyticactivity, rose linearly with volume of the X-substituent, X =H, Cl, Br, CH3 CHZ, (CH 3 )2 CH, (CH 3 ),S+, (CH 3 ) 3 NW, (CH 3 ) 3 C; thetrimethyl subsite accommodates the larger (CH 3 )3s substituentwith no further increase in activity. fi-X-Substituents withpolar surfaces, CH3 S(0 2 ) and (CH 3 ),N+O- showed lower reactiv-ity than consistent with volume.

- - ....... ...... ..........

C .-E .,Lm.,

3

summary ~jT"•' This research seeks information about the properties and

amino acid content of the active site of acetylcholinesterase(AcChE). It is based largelylon the view that the subsite atwhich the positively charged k-substituent of the naturalsubstrate, acetylcholine (AcCh), binds i; not anionic, as isgenerally accepted, but uncharged and complementary to the tri-methyl substituted character of that substituent. The subsitewould be more specifically explored by uncharged reagents,while positive reagents would react at peripheral negativesites. Thus, uncharged irreversible and reversible inhibitorsand substrates are obtained or synthesized and their reactionswj_tbhthe enzyme are studied. Primary emphasis is placed on'labeling the active site with 1-14C-l-bromopinacolone, (CH 3 ) 3 C-C0 1 4CH Br (14C-BrPin); we have found in previous work thatBrPin inactivates AcChE with the same kinetics toward hydro-lysis of substrates of widely varied structure.

In the current contract year equipment was assembled andAcChE was isolated from Torpedo nobiliana by affinity chromato-graphy; gel electrophoresis showed a major band at -70 kD,other bands at 124 and 103 kD, quite faint bands at 33, 50 and45 kD. A sample was labeled with 3 H-diisopropylfluorophos-phonate (DFP) and showed radioactivity only at 66-67 kD. Syn-thesis of 14C-BrPin in micromole quantity will be worked on.Purchased Sigma eel AcChE was found to contain much extraneousprotein and degraded enzyme subunit, -50 kD. It is satisfac-tory for kinetic studies, with acetylcholine controls, butisolated Torpedo enzyme will be used for characterization.

Other potential irreversible inhibitors were surveyedbriefly in enzymic hydrolysis of AcCh. Chloromethyl pivalate,(CH3 ) 3 CCOzCH.CI, closely related to BrPin, inactivated to anextent similar to BrPin. Chloromethyl acetate, CHCO2CH2C1,which probably binds at the esteratic or acetyl subsite,appeared to act both as a weak substrate, -1% as reactive asAcCh, and as an irreversible inhibitor, similar in activity toBrPin. Trimethylammoniomethyl acetate, (CH 3)3N+CH; OCOCH 3 , thelower homologue of AcCh, acted as a reversible inhibitor, K1 -1 mM, as a substrate, -10% as reactive as AcCh, and as a weakirreversible inhibitor, possibly by alkylation or by formationof formaldehyde in the active site. tert-Butyl peracetate,(CH3 ) 3 COOCOCH3 , acted as both a substrate, -10% as reactive as

AcCh, and as an irreversible inhibitor, possibly by generatingtert-butyl hydroperoxide, (CH3) 3 COOH, in the active site.

"The methanesulfonyl group, CH3 S(O 2 ), similar in shape totert-butyl, (CH0 3 )C, appears to lead to similar binding whenexamined in methylsulfonylethyl acetate, CH3 S(O 2 )CH2 CH2 OCOCH 3 ,both as a substrate and as E reversible inhibitor. In methylmethanesulfonate, CH 3 SO2 OCH 3 , it leads to rapid irreversibleinhibition, probably by alkylation; methylsulfonyl chloride,

4

CH3 SOZC1, inactivates like methylsulfonyl fluoride, probably byacylating the serine hydroxyl. The CH3 SO, group in reversibleinhibitors may lead to useful water and oil permeable modera-tors of AcChE activity.

The trichloromethyl group in trichloroethanol, C1 3 CCH2 OH,leads to weak binding similar to that of its carbon analogue,neopentyl alcohol, (CH 3 ) 3 CCH2 OH, K, = -40 and 30 mM, respec-tively. Chloral hydrate, Cl CCH=O.HO, KI = I mM, binds morestrongly than its carbon analogue, pivalaldehyde, (CH3) 3 CC1=O.

Examination of an aryl binding subsite was begun. Methylbenzenesulfonate, C6 H5 SO'OeCH3 , K. = 6 mM, rapidly inactivatesirreversibly, and this inactivation is not retarded by tetra-methylammonium ion. Styrene epoxide, K1 = 10 mM, inactivatesAcChE less rapidly. Nitrobenzene is an effective reversibleinhibitor, K, = 0.5 mM, in hydrolysis of AcCh, while, likesmall cations, it accelerates hydrolysis of ethyl acetate.Further studies will be carried out on the charge-transfercharacter of the aryl binding subsite, and on development oflabeling agents for it.

A study of hydrolysis of p-substituted-ethyl acetates, 8-X-CH2 CH2 OCOCH3 , by AcChE was completed and published. Reactiv-ity, normalized for effect of the fl-substituent on intrinsichydrolytic reactivity toward hydroxide, log (ka,t/Ký ),, riseslinearly with volume of the substituent, as measured by molarrefractivity (MR) for f-X = H, Cl, Br, CH3 CH., (CH 3 ) 2 CH,(CH3 ) 2 S+, (CH 3 ) 3 N+ and (CH3 ) 3 C; reactivity levels off with thelatter, while the trimethyl site accommodates the larger(CH3 ) 3 Si group with no further increase in reactivity. fi-Substituents with polar surfaces, CH 3 S(02) and (CH3 )2 N+O , showlower reactivity than is consistent with their MR values,possibly because they do not remove H20 from the active site.The correlation with MR is superior to that with hydrophobi-city, w, which predicts low reactivity for the importantpositively charged substrates. Study of substrates willprobably not be extended.

* Collaboration continues with Professor J. B. Cohen,Department of Anatomy and Neurobiology, Washington UniversitySchool of Medicine, St. Louis. Compounds for his study of theAcCh receptor are prepared at Brandeis; labeling of AcChE iscarried out at Brandeis; and final characterization of labeledpeptides will be carried out at Washington University.

5

FOREWORD

Citations of commercial organizations and trade names in thisreport do not constitute an official Department of the Armyendorsement or approval of the products or services of theseorganizations.

----- =3

,?~

6

Table of Contents

Page

Summary 3

Foreword 5

ReportStatement of the Problem Under Study 7Background and Review of Appropriate Literature 7Rational.? for the Current Study 9Experimental Methods

Isolation of Acetylcholinesterase 9Kinetic Procedures 11

ResultsIsolation and Labeling of AcChE 12Table I. Isolation of Torpedo AcChE 12Other Potential Irreversible Inhibitors 13Reversible Inhibitors 14Benzene Derivatives as Inhibitors and

Accelerators 15Substrates 16

Discussion and Conclusions 16Table II. Hydrolysis of X-CH2CH2 OCOCH3 by

Acetylcholinesterase, pH 7.8, O.18M NaCl, 25"CMolar Refractivity (MR), Hydrophobicity (r),and Kinetic Constants 20

Figure 1. Normalized Reactivity of X-CH2CH2OCOCH 3 ,iog(kc t/k )n and Mnlar Refractivity, MR,,-) 21

Figure 2. Normalizedd Reactivity of X-CHzCHOCOCH3 ,log (k,,t/k ),, and Hydrophobicity (r)(x 22

References 23

Distribution List 27

7

(1) Statement of the Problem Under Study

We seek information about the properties of the activesite of acetylcholinesterase (AcChE) by study of the responseof the enzyme to substrates and inhibitors of varied structure.The work is based on our view that the subsite at which thepositively charged group of acetylcholine (AcCh) binds is notanionic, but is uncharged and better considered trimethyl,complementary to the trimethyl property of that substituent.We consider that this subsite may be irreversibly labeled morespecifically by reaction with a trimethyl-substituted alkyl-ating agent, 1-bromopinacolone (BrPin), (CH 3 ) 3 CCOCH2 Br.

We have isolated AcChE from Torpedo nobiliana, purified itby affinity chromatography, labeled it with 3 H-diisopropyl-fluorophosphonate (DFP), subjected it to gel electrophoresis,staining and autoradiography. Distinct bands were found onstaining, and radioactivity was found at 66-67 kD. PurchasedSigma eel AcChE showed many protein bands on gel electrophore-sis and 3 H-DFP label at 49 and 52 kD. Purified Torpedo enzymewill be used in characterization of the active site; Sigmaenzyme may be used for kinetic studies.

Other active site-directed neutral and cationic irreversi-ble inhibitors are being developed: chloromethyl pivalate,(CH 3 ) 3 CCOCH.Cl; chloromethyl acetate, CH3 CO2 CH2 Cl; trimethyl-ammoniomethyl acetate, (CH3 )3N+ CH2 OCOCH3.; and tert-butylperacetate, (CH 3 )3 COOCOCH3 , potential alkylating agents and/orsuicide inhibitors. The trimethyl site was further exploredwith inhibitors and substrates containing substituents similarto trimethyl groups in shape and volume but differing insurface properties, with Cl and 0 replacing methyl: methylmethanesulfonate (MMS), CH3SO2 OCH3 , methylsulfonyl chloride(MSC), CH3S02Cl, methylsulfonylethyl acetate, CH3 SO2CH2CHMO-COCH3 , trichloroethanol, Cl 3 CCH7OH, and chloral hydrate,Cl 3 CCH=0.H.0. These compounds, except the last three, may beuseful labeling agents. BrPin remains preferred at present forspecific study of the trimethyl subsite, and will be preparedwith "C-label. Benzene derivatives were studied briefly asreversible and irreversible inhibitors of AcChE; effects ofelectron donating and withdrawing substituents will be studiedfor information as to the nature of the binding interaction atan aryl binding subsite.

(2) Background and Review of Appropriate Literature

Although the part of the active site of AcChE binding thetrimethylammonium group of AcCh, (CH3)3 NCH2 CH2 OCOCH3 , hasgenerally been considered as anionic (1-3), our recent studies

indicate that it may not contain a specific negative charge,and may be better considered trimethyl, as complementary to thetrimethyl-substituted character of the f-substituent ratherthan to its positive charge (4). This was proposed when theenzymic reactivity of a series of p-substituted ethyl acetates,X-CH2 CH2OCOCH3 , with cationic, uncharged polar and non-polarsubstituents was satisfactorily accounted for by two propertiesof the R-substituents: (i) their effects on the intrinsic reac-tivity as measured by hydrolysis by hydroxide, and (ii) theirapparent molar volumes, V!5. This is indicated in eq. 1,

log (k2 /K. aV. + C (1)

where log (k2c, /K ) is the enzymic reactivity normalized forintrinsic hydrolytic reactivity. Normalized reactivity, indi-cated by k-1• or (k /K),, is the experimentally observedvalue multiplied by the ratio of rate constants for hydrolysisof AcCh by OH" to that for the acetate substrate being studied.The effect of cationic charge in X is to increase intrinsic re-activity by a factor of about 25 as compared with that foranalogous alkyl substituents, and substantial effects of thepostulated (5) specific anionic Site in attracting and orient-ing cationic substrates are not required (4).

The enzyme has isoelectric point -pH 5 (6,7), and thus hasexcess negative charge on its surface at pH 7-8. Cationic re-versible inhibitors structurally related to AcCh bind betterthan their uncharged analogues by small factors, correspondingto -1 kcal/mole of binding energy, much less than would becaused by the interaction of (CH 3 ) 3 N with a "contact" anionic0- that is implied by a specific anionic site (8). Moreover,ionic strength effects on binding and on hydrolysis have been"interpreted in terms of anionic charges on the enzyme surfaceperipheral to the active site (9). Each of the inhibitorsstudied was structurally related to AcCh and, whether neutralor cationic, showed essentially the same binding constant whenretarding hydrolysis of AcCh and its uncharged analogue, 3,3-dimethylbutyl acetate. This indicated that the 0-trimethyl-ammonio and 9-tert-butyl groups of the two substrates and ofthe related inhibitors bind at the same subsite (8). Thesubstrate study had indicated that this subsite is complementa-rxy to uncharged trimethyl, and the properties of the subsitewould then be more specifically explored with unchargedreagents (4). Indeed, arylaziridinium reagents, intended toalkylate the "anionic" site and prevent substrate access,

2 inhibited completely the hydrolysis of cationic but not ofneutral esters (10,11). We took this to indicate not that"there are separate anionic and neutral subsites, but thatcationic agents act at peripheral anionic groups, thus increas-ing positive charge and repelling cationic substrates andinhibitors; they modify the active site domain but allow

I

9

uncharged substrates to bind at the one trimethyl site andreact. This view was borne out in studies with 1-bromopina-colone, (CH 3 ) 3 CCOCH2Br (BrPin), which inhibited AcChE irrevers-ibly with the same effect on hydrolysis rates of a variety ofboth cationic and neutral substrates. BrPin apparently exertedits inhibitory effect via binding at the trimethyl site (12).

(3) Rationale For the Current Study

The results indicating that AcChE inactivation by BrPinappears to involve BrPin interaction with the active sitesuggest these studies to explore the AcChE active site:

(i) isolation and labeling of AcChE from Toredo,nobiliana; receptor studies are carried out with electric organfrom Torpedo nobiliana at Brandeis and at Washington Universi-ty, and frozen organ is readily available locally.

(ii) a program to characterize the active site bylabeling amino acid(s) in it with radioactive BrPin,

(iii) attempts to prepare other irreversible inhibitors,potential labeling reagents based on the tert-butyl group,

(iv) exploration of the active site with acetatesubstrates containing varied f-substituents -- (CH3 )3Si-,(C1 3 )C-, (CH 3 ) 2S-, CH3 S(0 2 )-, (CH 3 )S(O)-, (CH 3 ) 2 N(O-)- -- andwith structurally related reversible and irreversible inhibi-tors,

(v) study of uncharged benzene derivatives as reversibleand irreversible inhibitors, and as accelerators.

(4) Experimental Methods

Isolation of Acetyicholinesterase.

Frozen electric organ of Tor•ogd nobiliana was obtainedfrom Sal Testaverde, Gloucester, Mass., and stored at -700C.Literature procedures for AcChE isolation were adapted (7,13)with purification by affinity chromatography (14,15).

The affinity ligand was prepared in accordance with lite-rature procedures (14,15) by the reaction series: acylation of6-aminohexanoic acid with benzyl chloroformate, condensation ofthis product with m-aminodimethylaniline in the presence of di-cyclohexylcarbodiimide MDCC), quaternization with methyliodide, and cleavage of the carbobenzyloxy group with HBr, eqs.2-5.

.1

I~0

H2 N(CH2 )5 C0 2 H + C6 HSCH2 OCOCl - C6 H.CH2 OCONH(CH.) 5 CO2 H (2)

C6 H 5 CH2OCONH(CH 2 ),CO2 H + m- (CH3 ) NC6 H4 NHZ

SDCC(3)

m- (CH3 ) NC6 H, NHCO (CH. ) 5 NHCO. CH C6 HS

+ CH3 1 (4)

m- (CH 3 NW C6 H4 NHCO (CH2 ) 5 NHC0 2 CH2 CS H5

+ HBr (5)

m- (CH3 )3 N- CS H NHCO (CH 2 ) 5 NH2 "HBr

The ligand ' coupled to 6-aminohexanoic acid-Sepharose B by1-(3-dimei: '3] ]minopropyl-3-ethyl)carbodiimide. The affinitycolumn was "isted with Sigma electric eel AcChE, which wasbound satis.actorily and eluted with 1 mM decamethonium; 120units were applied to the column and 99 units were recovered.Enzyme was assayed by the Ellman procedure (16).

Sodium dodecyl sulfate (SDS) polyacrylamide gel equipmentwas assembled and procedures for assay of AcChE labeled with3 H-DFP were carried out (17,18). Slab gels were 8% polyacryl-amide, 1.5 mm thick; stacking gels were 4% polyacrylamide.Electrode buffer contained 0.04 M Tris, 0.38 M glycine, 0.1 mMsodium thioglycolate and 1.5 g/L SDS. This composition led topH 8.3 and no additional buffer was used. Staining solutionwas 1.25 g Coomassie blue in 500 mL of 1:1:0.2 methanol,distilled water, acetic acid. Destaining solution was 25%methanol, 10% acetic acid in distilled water. Staining anddestaining were carried out at room temperature. Sigma SDSmarkers were used: myosin, M.W. 205,000; A-galactosidase,116,000; phosphorylase b, 97,000; bovine albumin, 66,000; eggalbumin, 45,000; and carbonic anhydrase, 29,000.

Labeling and electrophoresis were examined with SigmaAcChE. An initial large amount was taken, since the enzymecontent appeared low: 620 pg in 150 pL of buffer I (0.1 MNaCl, 40 mM MgCI 2 , 10 mM NaHCO3 , pH 8.0) was divided into 2portions; to 1 was added 2 AL of 50 mM decamethonium, and bothwere treated with 30 pL of 5.7MM (0.9 pCi) of 3 H-DFP at 4VC forI hr. p-Mercaptoethanol, 20 AL, was added to 1 mL of a stocksolution of 40% sucrose, 10% SDS, 0.3 M Tris (pH 6.8), 0.0125%bromophenol blue, and 2% glycerol; 25 ML of this was added to105 ML of the enzyme solution, and this was heated at 100"C for

m1'1:1! 1 111 A I

'iII1171 11'N m

2 min. Portions of the dissociated enzyme were loaded onto thestacking gel and electrophoresis was carried out at 100 V for5-6 hr, with the markers on a separate track. The gel wasprefixed for 20 min in a solution of 50% methanol, 10% aceticacid, and 40% distilled water, stained with Coomassie blue for30 min, destained overnight, treated with Enhance for 1 hr andwith distilled water for 1 hr and dried in a Biorad gel dryerfor 2 hr at 68"C. Stain bands were found at 81, 73, 52, 49,37, and 29 kD. Streaking of protein on the gel was observed.The Sigma AcChE, used as received for examination of the label-ing and electrophoresis, appeared to contain a range of proteinfractions. The gel was placed in contact with preexposed KodakX-Omat film for 43 hr at -700C and developed. The solutiontreated with 3 H-DFP in the presence of decamethonium showed nolabel; the one without decamethonium showed 3 H-DFP label at 52and 49 kD. The active fraction, reacting with DFP, appearedsomewhat degraded, as compared with an expected subunit of -70kD. The equipment and procedure appeared satisfactory andattention was turned to isolation and purification of AcChE.

Electric organ tissue, -500 g, was thawed at 40C,suspended in -1500 mL of buffer I, homogenized for four 1minute periods at 4"C and centrifuged for 2 hr at 7000 rpm in a6 x 250 mL rotor. The pellet was resuspended in 250 mL ofbuffer I, treated with 5 pg/mL of Sigma trypsin for 10 min at37'C, and then with 10 Ag/mL of Sigma Type I-S soybean trypsininhibitor. The suspension was cooled to VC, centrifuged for90 min at 25 K in a 12 x 25 mL rotor, applied to a 12 x 1.5 cmaffinity column, and eluted at 37 mL/hr, VC. The column waswashed with buffer I until clear of protein and assayed at 280nm. Enzyme was eluted with 1 mM decamethonium in buffer I;protein and activity were measured (16). Active fractions werepooled and decamethonium was removed on a 15 x 1.5 cm C-2-120CM Sephadex column; the eluate was concentrated to 5 mIL byultrafiltration, dialyzed against three 1 L portions of bufferI for 48 hr, further concentrated to 450 AL (Amicon micro-ultrafilter) and stored at -70"C in 100 AL aliquots. Theconcentrated solution was assayed for protein with a Bioradassay kit and for enzyme content by hydrolysis of acetyithio-choline (16). All steps were carried out at 4C uniess other-wise noted.

Kinetic Procedures.

Kinetic studies were carried out with pH stat equipment asdescribed in our previous publications (4,8). Exploratorystudy of potential irreversible inhibitors was carried out asin the study of inhibition by BrPin (12). Inhibitorscontaining acetate ester functions were also examined brieflyas substrates (4). Reversible inhibitors were studied asdescribed previously (8).

7.

12

(5) Results

Isolation and Labelinic of AcChE.

-9 Affinity ligand and an affinity column were prepared andtested. Gel electrophoresis equipment was assembled and testedwith 3 H-DFP-labeled Sigma eel AcChE. Frozen electric organ ofTorpedo nobiliana was obtained; three 500 g portions were usedfor enzyme isolation. AcChE was obtained in concentratedsolution by affinity chromatography and decamethonium elution.

Table I

Isolation of Torpedo Acetylcholinesterase

Volume AcChE Units Protein AcChEjamole

Preparation ;L AcSCh/mina mg unit/mg

(1) 230 7860 6.44 1220b

(2) 320 9080 5.56 1650b

(3) 450 7800 4.18 1866

a AcSCh, acetylthiocholineb These samples contain decamethonium.

An aliquot of solution 3 above, 10 AL (93 Mg protein)diluted to 30 AL with phosphate buffer, pH 7, incubated with 8uL of 5.7 UM 3 H-DFP and subjected to electrophoresis and auto-radiography, showed a broad heavily stained band at 66-72 kD,other bands at 103 and 124 kD, and faint bands at 45, 50, and133 kD. Little stain was seen at the dye front, indicatinglittle degradation. Use of less protein will give betterresolution. 3 H-DFP label was observed only at the major, broadband, at 66-67 kD. The value of kca; for AcChE from Torpedoc lifornica (3.3 x 105 min-') (19), if valid for Tor,_edonobiliana, leads for solution 3, above, to (7.8 x 10-3)/(3.3 x105) = 2.4 x 10-8 mole of AcChE, equal to 1.6 mg (m.w. =67000). Thus, solution 3 contains 1.6 mg of AcChE in 4.18 mg ofprotein, or -40% enzyme.

In one solution, salt regeneration of the column afterelution with decamethonium led to recovery of additionalenzyme, 1270 units in 4.61 mg, 275 units/mg. This material

K. subjected to labeling and electrophoresis showed a minor band

Li4 1

13

at 99 and a major band at 73 kD, and only the latter waslabeled by 3 H-DFP.

Preliminary experiments were carried out on AcChE as itwas being isolated from Torpedo. Trypsin-released enzyme,before purification by affinity chromatography, was completelyinactivated by 3 mM BrPin in 4 hr, while Sigma eel AcChE was-50% inactivated. On the other hand, as a competitive reversi-ble inhibitor, BrPin had K, = 0.7 mM for Torpedo AcChE, 0.2 mMfor eel enzyme. 4-Oxo-N,N,N-trimethylpentaminium, (CH 3 ) 3 N+CH2 -CH2 CH2 COCH. , had K, = 0.2 mM for Torpedo, 0.08 mM for eelenzyme. Hydrolysis of AcCh led to N = 0.05 mM for Torpedo,0.3 mM for eel enzyme. Column-purified enzyme will becharacterized.

Other Potential Irreversible Inhibitors.

Chloromethvl 2ivalate, (CH3 ) 3 CCOOCH2 C1 (Aldrich), 3.5 mM,structurally analogous to BrPin, led to complete inactivationafter 3 hr when the incubating solution was allowed to becomeacidic; 50% inactivation was observed when pH was maintained at7.8. This compound, examined as a substrate, appeared tohydrolyze slowly.

Chloromethyl acetate, CHCO2 CH2 C1, was synthesized fromformaldehyde and acetyl chloride (20). At 4.4 mM it led to 90%inactivation after 4 hr when the incubation solution wasallowed to become acidic, and to 70% inactivation when pH wasmaintained at pH 7.8. As a substrate it showed k at / -104sec 1/16 mM = 6 x 10 M-i1 sec-1, 4 x 105 M-1 sec" whennormalized for its high intrinsic hydrolytic reactivity; k(OH-)- 4.4 M-1 sec"'.

No reactivation occurred in either case on standing or ontreatment with 2-methyl-pyridinium aldoxime (PAM).

Trimethylammoniomethyl acetate, (CH3 )3N9. CHOCOCH3 (21),examined as a reversible inhibitor, showed mixed competitive(comp) and noncompetitive (nonc) inhibition, KI(,COp) = 1.4 mM,

KI(n -nc = 4.8 mM. As a substrate it had kc / = 3.9 x 103sec-1/0.8 mM = 4.5 x 106 M" 1 sec-', 3.8 x 10 M-_ sec"' whennormalized for intrinsic reactivity; k(OH -) = 3.3 M-1 sec"'.After hydrolysis of 3 mM of this compound, reactivity of theenzyme was reduced -30%.

tert-Butyl Deracetate, (CH 3 ) 3 COOCOCH3 (Pfaltz and Bauer),is hydrolyzed by AcChE, k at/Km = 3.4 x l0 sec-1/2.8 mM = 1.2x 108 M1 sec-., 1.7 x i0• M-1 sec-1 when normalized for itsintrinsic hydrolytic reactivity; k 0O-) = 2.0 M-1 sec-1. Themeasured enzymic activity was greater than that of its carbonnon-peroxidic analogue neopentyl acetate, (CH 3 ), CCHOCOCH3 ,kct/N = 1.9 x 102 sec- 1/8.6 mM = 2.2 x 104 M- sec'1, butnormalized activities were similar, 8.8 x 10 M-1 sec-' for the

-$

14

neopentyl compound. Enzymic hydrolysis (4 riM enzyme) of 3 mMperacetate was complete in 1 hr, when enzymic reactivity wasdecreased -30%. Further additions of two portions of 3 mMperacetate led to 50 and 30% residual activity. Similar treat-ments with 2 mM tert-butyl hydroperoxide (Aldrich) led tosmaller decreases in reactivity, 10, 15 and 25%.

Methyl methanesulfonate (MMS), CH3 SO2 0CH3 , (Aldrich). Thisalkylating agent, at 9 mM, inactivated rapidly, 40% in 1.6 hr,completely in 4 hr, in hydrolysis of both AcCh and 3,3-di-methylbutyl acetate. At 1 mM no inhibition was observed for 4hr, then 50% inactivation was found after 6 hr. At 3 mM therewas 30% inactivation in 1 hr, 70% in 2 hr. Tetramethylammoniumion, 30 mM, and 4-oxo-N,N,N-trimethylpentaminium, 0.2 mM,afforded complete protection for 1 hr and then inactivationproceeded. As a reversible inhibitor, MMS acted purely non-competitively, K1 = 96 mM.

Methyl benzenesulfonate (MBS), C6H5 SO. OCH3 (Pfaltz andBauer), showed mixed reversible inhibition, KI(€ 0 , = 6 mM,K= 21 mM, and irreversible inactivation. At 3 mM,there was 65% inactivation in 1 hr, 95% in 2 hr, largelyunaffected by 30 mM tetramethylammonium ion. At 0.6 mM therewas no inactivation after 1 hr, 15% after 3.3 hr, 33% after 5hr, 93% after 24 hr, with similar effects in hydrolysis of bothAcCh and 3,3-dimethylbutyl acetate.

M•ethvlsulfonvl chloride (MSC), CH3 SOCI (Aldrich), at 3mM, inactivated AcChE completely in 10 min; at 0.3 mM, itinactivated 54% in 10 min, and inactivation was complete after19 hr. At 0.03 mM there was 38% inactivation after 10 min, 72%after 14 hr. MSC appears as active as methylsulfonyl fluoride(MSF), CH3 SOZF.

Reversible Inhibi.g__.

Methvlsulfonylethyl acetate, CH3 SO2 CH2 CH2 OCOCH3 (22),pieviously studied as a substrate, kc at/K = 1.1 x 103 sec"'/6.2 mM (23), was studied as a reversible inhibitor forhydrolysis of AcCh and found to have KI(comp) = 6.4 mM,

5-Dimethvlamino-2-pentanone-N-oxide, (CH 3 ) N (0-) CH2 C 2 -CH2 COCH3 (synthesized), shows competitive reversibleinhibition, Ki - 14 mM, very similar to binding of thecorresponding ester substrate, (CH3) 2N (0-) CH2 C 2 CH, OCOCH3 K= 18 mM (23).

Trichloroethanol, C1 3 CCH OH (Aldrich), K = 42 mM,KI2 (onc - 110 mM, inhibits similarly to its trimethyl ana-logue, neopentyl alcohol, (CH3 ) 3 CCH2 OH (Aldrich), KIC.oI ) = 29mM, KI(nonc) = 74 mM, and to the ether isomer of the lattr,

15

Smethyl-t-butylether, (CH3 ) 3 COCH3 (Aldrich), KIComP) = 25 mM,SKi(nonc) = 24 mM.

Chloral hydrate, Cl 3 CCH=0-H 2 0 (Aldrich), is an effectivecompetitive reversible inhibitor, KI = 1 mM, binding substan-tially more strongly than its trimethyl analogue, pival-aldehyde, (CH 3 ) 3 CCH=0 (Aldrich), K, = 10 mM.

In collaboration with Professor J. B. Cohen, WashingtonUniversity School of Medicine, St. Louis, the enantiomers ofdiethylaminoethvl a-phenylbutvrate, (+) and (-) CH 3 CH 2 CH(CB H5 )-COOCH2 CH. N (C2 H5 ) z, were prepared.

Addition of N,N-dimethyl- and N,N-diethylhydroxylamines tomethylvinyl ketone has not been successful:

RZ NOH + CH2 =CHCOCH3 #--* R2 NOCH 2 CH2 COCH 3 (6)

Addition of methanesulfonamide, which shows no reversibleinhibition, to methylvinyl ketone, was intended to form a 1:1adduct but appeared to form a 1:2 adduct which then cyclized:

CH3 SO2NH2 + 2CH2 =CHCOCH 3 CH3 SO2 N (CH2 CHCOCH3 )-

CH3

OzH S\ /CH2 (7)

N- CH2

CH2 CH. COCH 3

Benzene Derivatives as Inhibitors and Accelerators.

Stvrene eDoxide (Aldrich) showed mixed reversibleinhibition, Kic' Mp = 10 mM, Kinonc). = 33 mM. At 3 mM itcaused progressive irreversible inactivation, -50% in 6 hr, 90%in 23 hr.

Attempts to convert phenacyl bromide, CH 5 COCH.Br, to itsenol acetate by reaction with isopropenyl acetate failed.

Nitrobenzene, previously found to be an efficientreversible mixed inhibitor in the hydrolysis of AcCh, K1 = 0.5mM, appears to be slightly less effective in inhibitinghydrolysis of 3,3-dimothylbutyl acetate, KX = 0.8 =M, and stillless effective towards n-butyl acetate, 2.1 mM, and 2-methyl-sulfonylethyl acetate, 2.8 mM.

-...= . A A A itif. tl.Iah.Ak . - i AUU . t~.~AiL

i6

Nitrobenzene accelerates enzymic hydrolysis of -0.1 M ethylacetate by decreasing K,; 1 mM nitrobenzene doubles the rate,and 2 and 3 mM increase it fourfold. Acetophenone, 7 mM,accelerates hydrolysis of ethyl acetate threefold, while benzo-nitrile is less effective, 7 mM leading to 50% acceleration ofethyl acetate hydrolysis.

Substrates.

In an extension of the study of the range of applicabilityof eq. 1, the relation of enzymic reactivity to volume of Psubstituent, an alternative measure of volume, refractionvolume, MR (24-27), which is available for a broader range ofstructures, was substituted for apparent molal volume V 5 "Results, largely obtained prior to the current contract, aresummarized in Table II and Figure 1. The increase in normal-ized enzymic reactivity with volume leveled off with AcCh and3,3-dimethylbutyl acetate. The larger analogous silyl com-"pound, (CH 3 ) 3 SiCH2 CH 2 OCOCH 3 , was accommodated in the activesite with no substantial increase in reactivity. The methyl-sulfonyl, CH3 SO2; dimethylamine oxide, (CH3 ),NW (0"); methyl-sulfoxy, CH3 S(O); and methylthio, CH3 S substituents led tosubstantially lower reactivity than is consistent with MRvalues. In correlation with hydrophobicity (r), a structuralparameter widely used in drug design (24,28-31), thesesubstituents (except for methylthio) and the important cationicsubstituents (CH3 ),3 N and (CH 3 ) S÷, showed substantiallygreater reactivity than is consistent with r values (Figure 2).It is noteworthy that the hydrophilic CH3SO2 substrate,Compound IV (Table II), binds similarly to the hydrophobic(CH 3 ) 3 C substrate, Compound II A, K3 - -6 mM, withsubstantially lower kct and greatly lower normalized reac-tivity (23).

(6) Discussion and Conclusions

A source of electric organ of Torpedo nobiliana is athand, and procedures have been worked out for isolation ofAcChE from this tissue by affinity chromatography in yields of-10 mg/kg. This is less than but comparable to reported(7,13) yields from To redo californica. Purity of the enzymeappears satisfactory for labeling with BrPin. Sigma eel AcChEcontains much low molecular weight material. Preliminaryexperiments on crude o enzyme, not purified by chromato-graphy, indicate rapid inactivation by BrPin. Properties ofpurified enzyme, i.e., K. for AcCh and K, for standardinhibitors, will be evaluated. BrPin remains the preferreduncharged reagent for active site labeling. Conditions must beestablished for preparation of "C-iabeled BrPin in Amolequantity. Additional purification of the enzyme beforelabeling will also be considered.

17

Other irreversible inhibitors are being prepared andexamined. Chloromethyl pivalate, (CH 3 )3 CCOOCH.C1, is apotentially useful reagent. At 3.5 mM, at pH 7.8, it inactiv-ates with the same effectiveness as 3 mM BrPin, 50% in 3 hr.It is not clear at present whether greater apparent effective-ness when the solution is allowed to become acidic is due toless destruction of the inhibitor or lower stability of theenzyme at acid pH. This may be checked with proper control.Enzyme inactivated at pH 7.8 is not regenerated by PAM,indicating that the active site serine may not be acylated andthat alkylation by the chloromethyl group occurs, as with thestructurally analogous BrPin. Long standing at pH 7.8 does notregenerate activity, indicating that the alkylation may not befollowed readily by hydrolysis of the ester group. Thiscompound may bind at the trimethyl site as BrPin does, and inalkylating, react with the same or a different nucleophile; itmerits further study.

Chloromethyl acetate, CH 3 CO2 CH2 Cl, inactivates the enzymeat pH 7.8 with efficiency similar to that of BrPin and chloro-methyl pivalate, and also shows greater effectiveness when thesolution is allowed to become acid. The same uncertaintyobtains as for the pivalate. This compound, an acetate, islikely to bind at the esteratic subsite, and may alkylate anucleophile in that area. Alternatively, its enzymichydrolysis may liberate chloromethanol, and then HC1 andformaldehyde in the active site, eq. 8, which may inactivatethe enzyme.

CH3 CO2 CH2 Cl -• CH3 CO 2 H + HOCH 2 Cl - HCl + CH=2 0 (8)

Trimethylammoniomethyl acetate, (CH3 ) 3 N CH2 OCOCH3 , thelower homologue of AcCh, is a moderately active substrate, with-1/10 the reactivity of AcCh, and appears to inactivate theenzyme as it hydrolyzes. This also may be a "suicide"inhibitor, due to formation of formaldehyde in the active site:

(CH3 ) 3 NWCH 2 OCOCH 3 - CH3 C02 H + (CH3 ) 3 NCHz OH

(CH3 )3 N+H + CH2 =0 (9)

tert-Butyl peracetate, (CH 3 )3COOCOCH 3 , may also act as a"suicide" inhibitor. It has fairly high enzymic reactivity,-1/10 that of the natural substrate, and leads to partial in-activation as it hydrolyzes, apparently by formation of tert-butyl hydroperoxide, (CH 3 ) COOH, in the active site as productof its enzymic hydrolysis. This inactivation is more rapidthan that caused by treatment with tert-butyl hydroperoxide.tert-Butyl hypochlorite, (CH 3 ) 3 COCI, may merit comparison withthe hydroperoxide, but study of esters as potential "suicide"inhibitors would take precedence.

18

Considering the binding subsite for the p-substituent astrimethyl, rather than anionic, leads to the study of unchargedsubstrates and inhibitors, which may be tetrasubstituted, butthe substituents need not be methyl groups. Thus 2,2,2-tri-chloroethanol binds similarly to the trimethyl analogues, neo-pentyl alcohol and tert-butyl methyl ether, and chloral hydratebinds very well, an order of magnitude better than its tri-methyl analogue, pivalaldehyde. The binding subsite appears toaccommodate the spherical substituents with surface Cl groupsas well as those of similar volume with CH3 surface groups.That the chloral carbonyl favors binding is noteworthy; thereason is not clear and one would hesitate to propose anytransition-state analogy. Further, the core atom may be Siinstead of C, since the larger (CH 3 ),Si group is accommodatedby the active site and leads to reactivity similar to that ofthe (CH 3 ) 3 C compound.

The methylsulfonyl group, CH3 SO 2 , appears to lead tounusual properties and may be of special interest. As a f-substituent in the ester CH3 SO2 CH2 CH2 OCOCH3 (MSAc), it leads tobinding similar to that of the analogous CH3 compound, (CH 3 ),C-CH2 CH2OCOCH 3 , whether measured as a substrate or as a reversi-ble inhibitor for hydrolysis of AcCh. However, it is substan-tially less reactive as a substrate. We have attributed thisto low effectiveness in removing water from the substrate-enzyme interface in the active site (23). There may, however,be an additional reason. Methanesulfonyl fluoride (MSF),CH3 SO F, and related compounds with good leaving groupsinactivate AcChE by converting the serine hydroxyl to themethylsulfonyl ester (32), and this action of MSF is accel-erated by certain alkyl and aryl cations (33). If the cationaccelerates by binding at the trimethyl site, then the CH3SO2is binding at the acetyl subsite, near the serine hydroxyl.Indeed, the CH3S(O) group in sulfone and sulfoxide is verysimilar to CH3 C(O). If the cation binds at a peripheralanionic site, this conclusion does not follow, but it remainsreasonable. Thus the low reactivity of the sulfonyl substrateMSAc, and of the analogous sulfoxy substrate, Compound IV A(Table II), may arise from non-productive binding, with sulfoneor sulfoxy groups interchanged with the ester group in theactive slte. Similarly, the low reactivity of the amine oxidesubstrate Compound III (Table II), which has the same bindingefficiency as substrate and as reversible inhibitor, may alsobe due to non-productive binding. Binding of the dimethylamineoxide at the acetyl subsite may resemble that of the dimethyl-carbamyl group (34). However, in the three cases, CompoundsIII, IV and IV A, low reactivity may also be due to inefficientremoval of water from the active site.

The sulfonyl group may be of interest as leading to waterand lipid permeability and possibly to reversible inhibitorswith biological action as moderators of AcChE activity. Whilemethylsulfonyl halides and related compounds are thought to

19

esterify serine and, thus, not to be useful labeling agents forother parts of the active site, methyl esters of methane-sulfonic and benzenesulfonic acids are likely to be methylatingagents and thus potential active site-labeling agents. Thispossibility is being explored, and retardation or accelerationof their action by organic cations will be examined.

Acceleration by organic cations of reactions of AcChE isnot restriced to inactivation by rethylsulfonyl fluoride.Hydrolysis of small substrates, such as methyl, ethyl andpropyl acetates, may also be accelerated in this way (35).When the subsite was considered anionic, this acceleration wasattributed to binding of the cations there, thus organizing theactive site and rendering it more effective. If the subsite isuncharged, as we propose, uncharged reversible inhibitors oflimited size might then also accelerate. In work carried outbefore the period of this contract, we observed that the simplearomatic compounds nitrobenzene and acetophenone are effectivereversible inhibitors, comparable to small organic cations.Our present observation that nitrobenzene and acetophenoneaccelerate enzymic hydrolysis of ethyl acetate to the sameextent as small cations indicates that cationic charge is notnecessary, although the electron-attracting character of thesubstituents may be important for this effect, as it is for thebinding. Thus, again, evidence for a specific anionic bindingsite has not been found. However, it is not clear exactlywhere these aromatic compounds bind.

With this renewed interest in simple aromatic inhibitors,we have now also observed that styrene epoxide shows irreversi-ble inactivation. This offers an opportunity for developingnew reactive agents for labeling the active site.

20

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21

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23

REFERENCES

1. "The Cholinesterases of Human Blood. 11. The ForcesActing Between Enzyme and Substrate", Adams, D. H., andWhittaker, V. P. (1950) Biochim. B-ioyhys. &gt!4 4. 543-558.

2. "Studies on Cholinesterase. VII. The Active Surface ofAcetylcholine Esterase Derived from Effects of pH onInhibitors"? Wilson, 1. B., and Bergmann, F. (1950) J.Bil hM 185, 479-48g.

3. Michelson, M4. J., and Zeimal, E. V. (1973) Ace2tyjchojine,p. 13, Pergamon Press, New York.

4. "Hydrolysis by Acetyicholinesterase. Apparent MolalVolumes and Trimethyl and Methyl Subsites", Hasan, F.* B.,Cohen, S. G., and Cohen, J. B. (1980) J. Biol. Chem.255, 3898-3904.

5. "Acetylcholinesterase. XII. Further studies of BindingForces", Wilson, I. B. (1952) J. Oiol. Chem. 197, 215-225.

6. "Acetylcholinesterase. II. crystallization, AbsorptionSpectra, Isoionic Point", Leuzinger, W., Baker, A. L., andCauvin, E. (1968) Proc. Nati. Acad. Sci. U.S.A. S9, 620-623.

7. "Acetyicholinesterase from To~eo Characterization ofan Enzyme Species Isolated by Lytic Procedures", Taylor,P., Jones, J. W., and Jacobs, N. M4. (1974) i!IQ.LPharmacoj._ 10, 78-92.

8. "Cationic and Uncharged Substrates and ReversibleInhibitors in Hydrolysis by Acetylcholinesterase(EC 3.1.1.7). The Trimethyl Subsite", Hasan, F. B.,Elkind, J. L., Cohen, S. G., and Cohen, J. B. (1981)J. Biol. CheM. 256, 7781-7785.

9. "Effective Charge on Acetyicholinesterase Active SitesDetermined from the Ionic Strength Dependence ofAssociation Rate Constants with Cationic Ligands", Nolte,H.-J., Rosenberry, T. L., and Neumann, E. (1980)

B.ocemistry 19, 3705-3711.

10. "The Properties of Acetyicholinesterase Modified byInteraction with the Alkylating Agent N,N-Dimethyl-2-Phenylaziridinium Ion", Purdie, J. E. (1969) Bioc im.Biophys. Act4 185, 122-133.

24

11. "A Novel Irreversible Inhibitor of AcetylcholinesteraseSpecifically Directed at the Anionic Binding Site:Structure-Activity Relationships", Belleau, B., and Tani,H. (1966) Mol. Pharmacol. 2, 411-422.

12. "1-Bromopinacolone, an Active Site-directed CovalentInhibitor for Acetylcholinesterase", Cohen, S. G.,Lieberman, D. L., Hasan, F. B., and Cohen, J. B. (1982)J. Biol. Chem. 257, 14087-14092.

13. "Major Component of Acetylcholinesterase in ToElectroplax Is Not Basal Lamina Associated", Viratelle,0. M., and Bernhard, S. A. (1980) D1hZ 19, 4999-5007.

14. "Purification of Acetylcholinesterase by AffinityChromatography and Determination of Active SiteStoichiometry", Rosenberry, T. L., Chang, H. W., and Chen,Y. T. (1972) J. Biol. Chem., 247, 1555-1565.

15. "Rapid and Complete Purification of Acetylcholinesterasesof Electric Eel and Erythrocyte by Affinity Chromato-graphy", Berman, J. D., and Young, M. (1971) RNatl. Acad. Sci. U.S.A. 68, 395-398.

16. "A New and Rapid Colorimetric Determination of Acetyl-cholinesterase Activity", Ellman, G. L., Courtney,K. D., Andres, Jr., V., and Featherstone, R. M. (1961)Biochem. Pharmaco!. 7, 88-95.

17. "Cleavage of Structural Proteins during the Assembly ofthe Head of Bacteriophage T4", Laemmli, U. K., (1970)Nature, 227, 680-685.

18. "Maturation of the Head of Bacteriophage T4. I. DNAPackaging Events", Laemmli, U. K. and Favre, M. (1973)J. Mol. Biol. 80, 575-599.

19. "Functional Consequences of Ligand-Dependent Conforma-tional Changes in Trypsin-Solubilized and in MembraneParticle Constrained Acetylcholinesterase", Pattison, S.,and Bernhard, S. (1978) Proc. Natl. Acad. Sci, U-.S.A. 75,3613-3617.

20. "The Reaction Between Acid Halides and Aldehydes. III.",Ulich, L. H., and Adams, R. (1921) J. Am. Chem. Soc. 43,660-667.

21. "The Soft Drug Approach", Bodor, N. (1984) CHEMTECH.28-38.

25

22. "Studies on Biological Methylation. Part XII. A Precursorof the Dimethyl Sulphide Evolved by PolysiDhoniafast:tigita. Dimethyl-2-carboxyethylsulphonium Hydroxideand its Salts", Challenger, F., and Simpson, M. I. (1948)J. Chem. Soc. 1591-1597.

23. "Effects of Volume and Surface Property in Hydrolysis byAcetylcholinesterase. The Trimethyl Site", Cohen, S. G.,Elkind, J. L., Chishti, S. B., Giner, J.-L. P., Reese,H., and Cohen, J. B. (1984) J. Med. Chem. 27, 1643-1647.

24. Hansch, C., and Leo, A. (1979) Substituent Constants forCorrelation Analysis in Chemistry and Biology, Wiley, NewYork.

25. "Aromatic Substituent Constants for Structure-ActivityV Correlations", Hansch, C., Leo, A., Unger, S. H., Kim,

K. H., Nakaitani, D., and Lien, E. J. (1973) J. Med. Chem.16, 1208-1216.

26. "The Physico-Chemical Approach to Drug Design andDiscovery", Hansch, C. (1981) Drug DeveloDment Research1, 267-309.

27. "Physical Properties and Chemical Constitution. Part XXI.Aliphatic Thiols, Sulphides, and Disulphides", Vogel,A. I. (1948) I. Chem. Soc. 1820-1825; "Part XXII. SomePrimary, Secondary, and Tertiary Amines", 1825-1833; "PartXXIII. Miscellaneous Compounds. Investigation of the So-called Co-ordinate or Dative Link in Esters of Oxy-acidsand in Nitro-paraffins by Molecular RefractivityDeterminations. Atomic, Structural, and Group Parachorsand Refractivities", 1833-1854, and preceding papers.

* 28. "Partition Coefficients and Their Uses", Leo, A., Hansch,C., and Elkins D. (1971) Chem. Rev. 71, 525-616.

29. "Quantitative Correlations Between Chemical Structure andAffinity for Acetylcholine Receptors", Lien, E. J.,Ariens, E. J., and Beld, A. J. (1976) European J.PharMacol. 3S, 245-252.

30. "Novel Hydrophobic Parameter for Ure in Structure-Activity Relation (SAR) Studies", Rekker, R. F., and Nys,G. G. (1974) in Molecular and Quantum PharmacoloQy,(Bergmann, E., and Pullman, B. Eds.) Reidel, Dordrecht-Holland, 457-463.

31. "Substituent Constants for Aliphatic Functions Obtainedfrom Partition Coefficients", Iwasa, J., Fujita, T., andHansch, C. (1965) J. Med. Chem. 8, 150-153.

26

32. "Esters of Methanesulfonic Acid as Irreversible Inhibitorsof Acetylcholinesterase", Kitz, R., and Wilson, I. B.(1962) J. Biol. €=,. 237, 3245-3249; "The Reactivation ofAcetylcholinesterase after Inhibition by MethanesulfonicAcid Esters", Alexander, J., Wilson, I. B., and Kitz, R.(1963) Biol. Chem. 238, 741-744.

33. "Acceleration of the Rate of Reaction of MethanesulfonylFluoride and Acetylcholinesterase by Substituted AmmoniumIons", Kitz, R., and Wilson, I. B. (1965) J. Biol. Chem.238, 745-748.

34. "Inhibition of Esterases by the Fluorides of OrganicAcids", Myers, D. K., and Kemp, A. (1954) Nature, 173,33-34; "Carbamylation of Acetylcholinesterase", Wilson,I. B., Hatch, M. A., and Ginsburg, S. (1960), J. Biol.Chem. 235, 2312-2315.

35. "Catalysis by Acetylcholinesterase. Acceleration of theHydrolysis of Neutral Acetic Acid Esters by CertainAromatic Cations", Barnett, P., and Rosenberry, T. L.(1977) J. Biol. Chem. 252, 7200-7206.

I.'

27

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