Solvent Deuterium Oxide Isotope Effects on the Reactions Of

molecules

Article Solvent Deuterium Oxide Isotope Effects on the Reactions † of Organophosphorylated Acetylcholinesterase

Terrone L. Rosenberry Mayo Clinic College of Medicine, Departments of Neuroscience and Pharmacology, Jacksonville, FL 32224, USA; [email protected] ; Tel.: +1-904-953-7375; Fax: +1-904-953-7370 This manuscript was given as an oral presentation on December 9, 2019 at the 16th Symposium on Cholinergic † Mechanisms in Rehovot, Israel. Academic Editor: Zrinka Kovarik Received: 1 August 2020; Accepted: 17 September 2020; Published: 25 September 2020 Abstract: Organophosphates (OPs) are esters of substituted phosphates, phosphonates or phosphoramidates that react with acetylcholinesterase (AChE) by initially transferring the organophosphityl group to a serine residue in the enzyme active site, concomitant with loss of an alcohol or halide leaving group. With substituted phosphates, this transfer is followed by relatively slow hydrolysis of the organophosphoryl AChE, or dephosphorylation, that is often accompanied by an aging reaction that renders the enzyme irreversibly inactivated. Aging is a dealkylation that converts the phosphate triester to a diester. OPs are very effective AChE inhibitors and have been developed as insecticides and chemical warfare agents. We examined three reactions of two organophosphoryl AChEs, dimethyl- and diethylphosphorylated AChE, by comparing rate constants and solvent deuterium oxide isotope effects for hydrolysis, aging and oxime reactivation with pralidoxime (2-PAM). Our study was motivated (1) by a published x-ray crystal structure of diethylphosphorylated AChE, which showed severe distortion of the active site that was restored by the binding of pralidoxime, and (2) by published isotope effects for decarbamoylation that decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE. We previously reconciled these results by proposing a shift in the rate-limiting step from proton transfer for the small carbamoyl group to a likely conformational change in the distorted active site of the large carbamoyl enzyme. This proposal was tested but was not supported in this report. The smaller dimethylphosphoryl AChE and the larger diethylphosphoryl AChE gave similar isotope effects for both oxime reactivation and hydrolysis, and the isotope effect values of about two indicated that proton transfer was rate limiting for both reactions.

Keywords: acetylcholinesterase; paraoxon; dephosphorylation; reactivation; D2O isotope

1. Introduction Acetylcholinesterase (AChE) catalyzes the hydrolysis of the neurotransmitter acetylcholine, and rapid hydrolysis of this ester is essential for normal cholinergic synaptic transmission. Acetylcholine hydrolysis proceeds by transfer of the acetyl group to the active site serine of AChE followed by hydrolysis, or deacetylation, of the acetyl enzyme, and both steps occur on a timescale of microseconds [1]. Other ester substrates of AChE proceed through a similar two-step catalytic pathway (Scheme1). However, the pathways for the esters in Scheme1 di ﬀer from that of acetylcholine in that these esters initially form a detectable reversible equilibrium complex with AChE with a dissociation constant, KD, before transfer of their carbamoyl or organophosphoryl groups (here referred to as acyl groups) to the active site serine to give carbamoyl or phosphoryl intermediates. This complex is an intermediate with acetylthiocholine but is not detectable, as explained in Equation (1) below. While the hydrolysis of the carbamoyl or organophosphoryl intermediates involves transfer of their

Molecules 2020, 25, 4412; doi:10.3390/molecules25194412 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 14 Molecules 2020, 25, x FOR PEER REVIEW 2 of 14 intermediates.Molecules 2020, 25 ,This 4412 complex is an intermediate with acetylthiocholine but is not detectable,2 ofas 14 explainedintermediates. in Equation This complex (1) below. is an While intermediate the hydrolysis with acetylthiocholine of the carbamoyl but or isorganophosphoryl not detectable, as intermediatesexplained in involvesEquation transfer (1) below. of theirWhile acyl the group hydrolysis from serineof the tocarbamoyl water, their or organophosphoryldeacylation rate constantsacylintermediates group (k3 from or involveskH serine) are orders to transfer water, of magnitude theirof their deacylation acyl smaller group rate than from constants that serine of acetylated (k 3toor water,kH) areAChE their orders [2,3]. deacylation of Their magnitude slow rate deacylationsmallerconstants than ( kmakes3 thator kH of )them are acetylated orderspotent of AChE inhibitors magnitude [2,3]. of Their AChE,smaller slow andthan deacylation some that ofcarbamates acetylated makes them and AChE potentorganophosphates [2,3]. inhibitors Their slow of (alsoAChE,deacylation denoted and some makesOPs) carbamates have them been potent widely and inhibitors organophosphates used as of insecticides AChE, (alsoand [4]. some denoted carbamates OPs) have and been organophosphates widely used as insecticides(also denoted [4]. OPs) have been widely used as insecticides [4].

KD k2 k3 E + CX KD ECX k2 EC k3 E + COH E + CX ECX EC E + COH + + X X

KD k2 kH E + OPX KD EOPX k2 EOP kH E + POH E + OPX EOPX EOP E + POH + + X X

Scheme 1. Two‐step catalytic pathway for carbamates (CX; [5,6]) and organophosphates (OPX; [7]). ProductsSchemeScheme 1. are1. Two-stepTwo the alcohol‐step catalyticcatalytic leaving pathwaypathway group X for forand carbamatescarbamates the carbamic (CX;(CX; acid [[5,6])5, 6(COH)]) andand or organophosphatesorganophosphates phosphate diester (OPX; (POH). [[7]).7 ]). ProductsProducts are are the the alcohol alcohol leaving leaving group group X X and and the the carbamic carbamic acid acid (COH) (COH) or or phosphate phosphate diester diester (POH). (POH). One noteworthy feature of AChE intermediates with carbamoyl and phosphoryl acyl groups (Figure One noteworthy feature of AChE intermediates with carbamoyl and phosphoryl acyl groups 1)One is that noteworthy their relative feature deacylation of AChE intermediatesrate constants with k3 or carbamoyl kH decrease and dramatically phosphoryl with acyl angroups increase (Figure in k k acyl(Figure1) is group that1) is theirsize. that relativeThis their decrease relative deacylation deacylation may result rate rate constantsin part constants from k3 or substantial 3kHor decreaseH decrease distortion dramatically dramatically of the with active with an site anincrease increasearising in frominacyl acyl groupthe group bulksize. size. Thisof This decreaselarge decrease acyl may may groups result result in in[8]. part part Forfrom from example,substantial substantial an distortion distortion X‐ray of crystal the active structure sitesite arisingarising of diethylphosphorylatedfromfrom the the bulk bulk of large of acyl hAChElarge groups acyl generated [8 ].groups For example,from [8]. paraoxon anFor X-ray example, [9] crystal is shown structurean in XFigure‐ray of diethylphosphorylated 2A.crystal In this structure structure, of thehAChEdiethylphosphorylated diethylphosphoryl generated from group paraoxonhAChE is attached generated [9] is shownto Ser203.from in paraoxon Figure One ethoxy2A. [9] In is group this shown structure, faces in Figure Trp86 the 2A.and diethylphosphoryl In the this other structure, faces Phe295,groupthe diethylphosphoryl is Phe297 attached and to Phe338 Ser203. group in One isan attached acyl ethoxy pocket. to group Ser203. Hydrogen faces One Trp86 ethoxy bonding and group the within other faces the faces Trp86 catalytic Phe295, and triadthe Phe297 other (Ser203 faces and‐ His447Phe338Phe295,‐Glu334) in Phe297 an acyl remains and pocket. Phe338 intact Hydrogen in but an theacyl bonding adjacent pocket. within acylHydrogen pocket the catalytic bondingresidues triad withinand (Ser203-His447-Glu334) acyl the loop catalytic (residue triad positions remains(Ser203 ‐ 280–297)intactHis447 but‐ Glu334)are the significantly adjacent remains acyl intactperturbed pocket but residues the relative adjacent and to acyltheiracyl loop pocketpositions (residue residues in positionsthe and ligand acyl 280–297)‐free loop hAChE (residue are significantly structure. positions Movementsperturbed280–297) are relative in significantlyresidue to theirbackbone perturbed positions positions relative in the are ligand-free to necessary their positions hAChEto avert in structure. stericthe ligand clash, Movements‐ freeand hAChE the Cα in structure.and residue the α sidechainbackboneMovements positionsof inArg296 residue are shift necessarybackbone by 4.9 topositions avertA° and steric are 14.9 necessary clash, A°, and respectively. to the avert C and steric Large the clash, sidechain acyl and loop the of Arg296 Cbackboneα and shift the rearrangementsbysidechain 4.9 A◦ and of 14.9 Arg296have A◦, respectively.shiftalso bybeen 4.9 Large seenA° acylandin loop14.9mAChE backbone A°, respectively.and rearrangements TcAChE Large inhibited haveacyl also loopby been thebackbone seen OP in diisopropylfluorophosphatemAChErearrangements and TcAChE have inhibited also [10,11]. bybeen the However, OPseen diisopropylfluorophosphate in stereoselective mAChE and inhibition TcAChE [10 ,of11 AChE]. However,inhibited by OP stereoselective nerveby the agents OP thatinhibitiondiisopropylfluorophosphate place a of sma AChE ller by methyl OP nerve group [10,11]. agents into However, that the place acyl stereoselective apocket smaller does methyl not inhibition groupaffect the intoof AChE acyl the acylloop by OP pocket in nerve the does crystal agents not structureathatffect place the in acyl athe sma loop same ller in manner themethyl crystal [9,10,12].group structure into the in theacyl same pocket manner does [not9,10 ,affect12]. the acyl loop in the crystal structure in the same manner [9,10,12].

FigureFigure 1.1. MolecularMolecular structures structures of carbamoyl of carbamoyl and phosphoryl and phosphoryl groups attached groups to human attached acetylcholinesterase to human acetylcholinesterase(AChE;Figure squiggled1. Molecular line) (AChE; comparedstructures squiggled in thisof line)carbamoyl report. compared Alkyl and groups in phosphorylthis R 1report.and R 2 Alkylgroupsare attached groups attached to R the1 and carbamoylto Rhuman2 are nitrogenacetylcholinesterase atom and alkoxy (AChE; groups squiggled R1 and line) R2 are compared attached toin thethis phosphoryl report. Alkyl phosphorus groups R atom.1 and R2 are

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Molecules 2020, 25, 4412 3 of 14 attached to the carbamoyl nitrogen atom and alkoxy groups R1 and R2 are attached to the phosphoryl phosphorus atom. These shifts in the acyl pocket residues and acyl loop in diethylphosphorylated hAChE can These shifts in the acyl pocket residues and acyl loop in diethylphosphorylated hAChE can be be largely reversed by cationic ligand binding to the active site [9]. The interaction of cationic largely reversed by cationic ligand binding to the active site [9]. The interaction of cationic oximes oximes with organophosphorylated AChEs is of intense interest because oximes have been shown to with organophosphorylated AChEs is of intense interest because oximes have been shown to reactivate these AChEs by displacing the serine hydroxyl and releasing the organophosphorylated reactivate these AChEs by displacing the serine hydroxyl and releasing the organophosphorylated oxime [7,13]. The structure in which 50 mM 2-PAM was diffused into the crystals to form a complex of oxime [7,13]. The structure in which 50 mM 2‐PAM was diffused into the crystals to form a complex diethylphosphorylated hAChE with the cationic oxime 2-PAM reveals two bound 2-PAM molecules of diethylphosphorylated hAChE with the cationic oxime 2‐PAM reveals two bound 2‐PAM (Figure2B), one in the peripheral site and the other stacked against the sidechain of Trp86. In this molecules (Figure 2B), one in the peripheral site and the other stacked against the sidechain of Trp86. ternary complex, the backbone conformation of the acyl loop resembles that of the ligand-free state In this ternary complex, the backbone conformation of the acyl loop resembles that of the ligand‐free and appears to be stabilized by oxime binding. Therefore, diffusion of these oximes into the crystals of state and appears to be stabilized by oxime binding. Therefore, diffusion of these oximes into the diethylphosphorylatedcrystals of diethylphosphorylated hAChE promoted hAChE promoted a restoration a restoration of the acyl of the pocket acyl residuespocket residues and acyl and loop acyl to theirloop locationsto their locations in the unmodified in the unmodified hAChE hAChE structure. structure.

FigureFigure 2. 2.Conformational Conformational changes changes in hAChE in hAChE induced induced by paraoxon by paraoxon inhibition [inhibition9]. Structural [9]. perturbations Structural seenperturbations in diethylphosphorylated seen in diethylphosphorylated hAChE without hAChE (A) and without with ( boundA) and 2-PAMwith bound (B) are 2‐PAM visualized (B) are by superimpositionvisualized by superimposition with ligand-free with hAChE. ligand Carbon‐free hAChE. atoms of Carbon the oximes atoms are of colored the oximes yellow, are and colored carbon atomsyellow, of and protein carbon residues atoms of are protein colored residues by complex: are colored green by in complex: (A) and green salmon in ( inA) ( Band); and salmon ligand-free in (B); hAChE,and ligand semitransparent‐free hAChE, gray.semitransparent Nitrogen, oxygen, gray. Nitrogen, and phosphorus oxygen, atomsand phosphorus are colored atoms dark blue, are colored red and orange,dark blue, respectively. red and orange, Residues respectively. of the helix Residues preceding of the the helix acyl looppreceding and of the the acyl acyl loop loop and up of to the position acyl 298loop are up drawn to position as ribbons. 298 are Atoms drawn of as select ribbons. residues, Atoms reactivators of select residues, and phosphate reactivators adducts and are phosphate drawn as sticksadducts and are hydrogen drawn as bonds sticks are and depicted hydrogen by bonds dashed are gray depicted lines. by dashed gray lines.

TheThe catalytic catalytic eeffectsffects ofof activeactive sitesite distortiondistortion in diethylphosphorylated AChE AChE on on the the deacylation deacylation pathwaypathway can be be examined examined by bydetermining determining D2O Disotope2O isotope effects eonffects the deacylation on the deacylation reactions. Here reactions. we Hereinvestigate we investigate the reaction the reactionrate constants rate constants and D and2O Disotope2O isotope effects eff ectsfor forthree three reactions reactions of of diethylphosphorylateddiethylphosphorylated AChE, AChE, which which shows shows crystallographic crystallographic evidence evidence of of active active site site distortion, distortion, and and of dimethylphosphorylatedof dimethylphosphorylated AChE, AChE, which which does does not not [9, 10[9,10,12].,12].

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Molecules 2020, 25, x FOR PEER REVIEW 4 of 14 2. Results 2. Results The primary objectives of this paper are to obtain the solvent D2O isotope effects for three The primary objectives of this paper are to obtain the solvent D2O isotope effects for three of four of four reactions that involve organophosphorylated AChEs. The four reactions are outlined in reactions that involve organophosphorylated AChEs. The four reactions are outlined in Scheme 2, Scheme2, and the isotope e ffects are shown in Table1 below. Because the determination of solvent D O and the isotope effects are shown in Table 1 below. Because the determination of solvent D2O isotope 2 isotopeeffects eff ectsrequires requires accuracy accuracy and andprecision, precision, an emphasis an emphasis is placed is placed on protocols on protocols for rate for constant rate constant measurement.measurement. TheThe isotope isotope eeffectsffects obtained are are then thencompared comparedto tothose those previously previously measured measured for for carbamoylatedcarbamoylated AChEs,AChEs, andand didifferencesfferences amongthem them are are interpreted interpretedin the in thecontext context of X‐ ofray X-ray crystal crystal structuresstructures determineddetermined forfor organophosphorylated organophosphorylated AChEs AChEs like like those those shown shown in Fig inure Figure 2. 2.

SchemeScheme 2. Four 2. Four reactions reactions of organophosphorylated of organophosphorylated AChE AChE (a –(ad–).d). The The alcohol alcohol RH, RH, the the phosphate phosphate diester diester R2P(O)OH, and the phosphate triester R2(oxime)P(O) are the products released during the R2P(O)OH, and the phosphate triester R2(oxime)P(O) are the products released during the aging, hydrolysisaging, hydrolysis and reactivation and reactivation reactions, reactions, respectively. respectively.

ProtocolsProtocols to to measure measure thethe rate rate constantsconstants of of the the reactions reactionsof oforganophosphorylated organophosphorylated AChEs AChEs in in Scheme 2, in both H2O and D2O, are outlined in the Materials and Methods, and representative Scheme2, in both H 2O and D2O, are outlined in the Materials and Methods, and representative reactions are illustrated in Figures 3–5 below. reactions are illustrated in Figures3–5 below.

2.1. Denaturation Rate Constants kX for Organophosphorylated AChE 2.1. Denaturation Rate Constants kX for Organophosphorylated AChE Before the rate constants for reactions b, c and d in Scheme 2 can be determined, the loss of AChE activityBeforearising the ratefrom constants enzyme fordenaturation reactions must b, c be and taken d in into Schemeconsideration.2 can be The determined, reaction denoted the loss of AChEdenaturation activity arising in Scheme from 2a enzymewas detected denaturationwith control mustAChE be and takenwith into fully consideration. reactivated AChE The in the reaction denotedabsence denaturationof OPs. Bovine in Scheme serum albumen2a was detected (BSA) was with added controlto the AChE assay solution and with to fullystabilize reactivated the AChE, AChE in thebut absence the enzyme of OPs. still showed Bovinea serumslow loss albumenof activity (BSA)with a was rate added constant tokX thettha assay(1) in solutionsome cases to was stabilize the AChE,close enough but the to the enzymeaging rate still constant showedkA aand slow the losshydrolysis of activity rate constant with a k rateH to require constant inclusionkX thatin (1) in the analytical equations and (2) was stabilized by 2‐PAM and thus depended on the concentration of some cases was close enough to the aging rate constant kA and the hydrolysis rate constant kH to −5 −1 require2‐PAM. inclusion Individual in the measurements analytical equations of kX ranged and from (2) 2–7 was × stabilized 10 min eand by 2-PAMar illustrated and thusin Figures depended 4 on the concentration of 2-PAM. Individual measurements of k ranged from 2–7 10 5 min 1 and are X × − − illustrated in Figures4 and5A below. The same denaturation constant kX was assumed to apply to free and organophosphorylated AChE. Molecules 2020, 25, x FOR PEER REVIEW 5 of 14 Molecules 2020, 25, 4412 5 of 14 and 5A below. The same denaturation constant kX was assumed to apply to free and organophosphorylated AChE. 2.2. Aging Rate Constants kA for Organophosphorylated AChE 2.2. Aging Rate Constants kA for Organophosphorylated AChE The fact that organophosphorylated AChE can undergo multiple reactions (Scheme2) required a sequentialThe fact approach that organophosphorylated to obtaining the rate AChE constants can undergo for these multiple reactions. reactions Fitting (Scheme some 2) of required the data herea sequential to equations approach required to obtaining preliminary the rate estimates constants of for some these rate reactions. constants Fitting that some were of refinedthe data inhere the fittingto equations of later data. required The preliminary aging reaction estimates in Scheme of some2b israte the constants loss of an that alkoxy were refined group attachedin the fitting to theof phosphoruslater data. atom The aging [14]. Thereaction resulting in Scheme phosphate 2b is the diester loss of that an alkoxy remains group covalently attached linked to the to phosphorus AChE is an extremelyatom [14]. stable The inactive resulting enzyme phosphate form diester that cannot that remains be reactivated covalently by linked oxime to (Figure AChE 3is). an The extremely protocol stable inactive enzyme form that cannot be reactivated by oxime (Figure 3). The protocol we used to we used to measure the aging rate constants kA is indicated in the Figure3 legend. We followed measure the aging rate constants kA is indicated in the Figure 3 legend. We followed this protocol to this protocol to obtain our best estimates of kA in both H2O and D2O. AChE was maintained in obtain our best estimates of kA in both H2O and D2O. AChE was maintained in a fully a fully organophosphorylated form for various times taged, after which it was diluted into a high organophosphorylated form for various times taged, after which it was diluted into a high concentration of 2-PAM to rapidly reactivate all of the enzyme that had not been aged. Figure3A concentration of 2‐PAM to rapidly reactivate all of the enzyme that had not been aged. Figure 3A demonstrates that, with reasonable initial estimates of kX, kA, kH, and kr, 2-PAM reactivation proceeds demonstrates that, with reasonable initial estimates of kX, kA, kH, and kr, 2‐PAM reactivation proceeds rapidly.rapidly. Fitting Fitting confirmed confirmed that that all all values values of ofv vmeasured measured after after thethe initialinitial value near tt == 00 essentially essentially v v v t correspondedcorresponded to the to the final final value value of of, denotedv, denotedF v.F Plots. Plots of of these these vFFagainst against tagedaged couldcould be be analyzed analyzed with with EquationEquation (3) (3) as shownas shown in in Figure Figure3B 3B to to give give values values of ofk AkApresented presented in Table1 1..

Figure 3. Aging of dimethylphosphorylated AChE in H2O. A mixture of 2.4 µM paraoxon methyl (OP) Figure 3. Aging of dimethylphosphorylated AChE in H2O. A mixture of 2.4 μM paraoxon methyl and 0.5 µM AChE in 100 mM sodium phosphate and 0.1% BSA (pH 8.0) was incubated for 60 min at (OP) and 0.5 μM AChE in 100 mM sodium phosphate and 0.1% BSA (pH 8.0) was incubated for 60 25 ◦C. The incubation was then aged for the various times indicated before addition of 2-PAM, with the min at 25 °C. The incubation was then aged for the various times indicated before addition of 2‐PAM, earliest addition of 2-PAM designated as taged = 0. To ensure that the OP remained in excess during with the earliest addition of 2‐PAM designated as taged = 0. To ensure that the OP remained in excess aging, additional OP was added (1.7 µM at taged 5 and 185 min and 3.6 µM at taged 425 min). At the during aging, additional OP was added (1.7 μM at taged 5 and 185 min and 3.6 μM at taged 425 min). At indicated t of 1, 180, 420 and 1470 min, portions of the aged reaction were diluted 24-fold into the indicatedaged taged of 1, 180, 420 and 1470 min, portions of the aged reaction was diluted 24‐fold into 1.0 mM 2-PAM. (A) Aliquots (30 µL) of each dilution were assayed as indicated in the Materials and 1.0 mM 2‐PAM. (A) Aliquots (30 μL) of each dilution were assayed as indicated in the Materials and MethodsMethods at the at timesthe times indicated indicated on the onx -axis.the x‐ Assayaxis. Assay values valuesv were v fit were to Equation fit to Equation (2), with (2), the with indicated the t fixed. Fixed initial estimates (min 1 for k values) of k = 2.5 10 5, k = 6.5 10 3, k = 9 10 2 agedindicated taged fixed. Fixed initial estimates− (min−1 for k values)X of kX =− 2.5 H× 10−5, kH = 6.5− × 10r −3, kr = 9 ×− 3 × × × and k −2 = 2 10 gave−3 fitted E = 0.86 ∆A/min (green line); this E was then fixed along with the 10A and kA = −2 × 10 gave fittedtot Etot = 0.86 ΔA/min (green line); this Etottot was then fixed along with the × 3 same values of k , k , and kr to fit k = 1–2 10 −3 and E = 0.01–0.02 ∆A/min (blue, red, and cyan lines). same values Xof kHX, kH, and kr to fitA kA = 1–2× × 10− and E00 = 0.01–0.02 ΔA/min (blue, red, and cyan lines). ValuesValues for timesfor times after after the initial the initial time pointtime point were observedwere observed to correspond to correspond to the finalto the value final of valuev, denoted of v,

vF, indenoted this equation. vF, in this (B equation.) Assay values (B) Assay corresponding values corresponding to vF in panel to AvF werein panel analyzed A were with analyzed Equation with (3) 5 1 6 1 withEquation a ﬁxed k (3)= with2.5 a 10fixedmin kX = 2.5to × give 10−5 Emintot −=1 to0.82 give∆A E/totmin = 0.82 and Δ aA/mink value and of a (980kA value+ 60) of (98010 + min60) × X × − − A × − − included10−6 min in− Table1 included1. in Table 1.

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2.3. Hydrolysis Rate Constants kH for Organophosphorylated AChE 2.3. Hydrolysis Rate Constants kH for Organophosphorylated AChE The hydrolysis rate constants kH were measured in H2O and D2O with the protocol in the Figure The hydrolysis rate constants kH were measured in H2O and D2O with the protocol in the Figure4 legend.4 legend. AChE AChE was was organophosphorylated organophosphorylated and dilutedand diluted with orwith without or without 200 µM 200 2-PAM. μM Assay2‐PAM. values Assayv fromvalues the v from dilution the withdilution 2-PAM with rapidly 2‐PAM reachedrapidly reached maximums maximums and were and analyzed were analyzed with Equation with Equation (2) to (2) to determine Etot. This value of Etot was then fixed in Equation (2) to fit the values of v from the determine Etot. This value of Etot was then fixed in Equation (2) to fit the values of v from the dilution dilution without 2‐PAM to obtain kH (Figure 4). without 2-PAM to obtain kH (Figure4). InIn contrast contrast to to the the protocol protocol for for the the aging aging reaction reaction in in Figure Figure3 A,3A, where where a a slight slight excess excess of of OP OP during during preincubationpreincubation was was rapidly rapidly turned turned over over by by dilution dilution into into a a highhigh concentrationconcentration ofof 2-PAM,2‐PAM, the the hydrolysis hydrolysis reactionreaction was was defineddefined inin thethe absenceabsence ofof 2-PAM2‐PAM andand itsits timetime coursecourse waswas sensitivesensitive to any excess of OP. An excess was indicated by an E0 close to zero and by a lag in the increase in v with time. An excess was indicated by an E0 close to zero and by a lag in the increase in v with time. Centrifugation throughCentrifugation a spin column through was a spin employed column towas minimize employed unreacted to minimize OP but, unreacted in addition, OP but, the in stoichiometry addition, the ofstoichiometry AChE and OP of AChE was set and close OP to was one set and close the to incubation one and the time incubation was limited time to was insure limited that, to in insure most that, in most cases, E0 was significantly greater than zero. However, this was not the case in Figure 4, cases, E0 was significantly greater than zero. However, this was not the case in Figure4, where E0 approachedwhere E0 approached zero. zero.

FigureFigure 4. 4.Denaturation Denaturation andand hydrolysis hydrolysis of of diethylphosphorylated diethylphosphorylated AChE AChE in in D D22O.O. AA mixturemixture ofof 2.02.0µ μMM

paraoxonparaoxon and and 1.4 1.4µ μMM AChE AChE in in 100 100 mM mM sodium sodium phosphate phosphate and and 0.1% 0.1% BSA BSA (bu (buffer)ﬀer) in in D D2O2O (pH (pH 8.0) 8.0) was was

incubatedincubated for for 70 70 min min at at 25 25◦ °C.C. A A 4-fold 4‐fold excess excess of of bu bufferffer in in D D22OO waswas added, added, and and 200 200µ μLL was was applied applied to to aa 0.5 0.5 mL mL Sephadex Sephadex G50 G50 spin spin column column (Fisher) (Fisher) that that had had been been prewashed prewashed four four times times in in bu bufferffer in in D D2O2O and centrifuged at 1000 g for 1 min. The eluent was cooled to 4 C, and 60 µL was added either and centrifuged at 1000×× g for 1 min. The eluent was cooled to 4 °C,◦ and 60 μL was added either to µ µ µ to640 640 μL ofL buffer of buff iner D in2O D at2O 25 at °C 25 or◦ C640 or μ 640L of theL ofsame the buffer same buwithffer 2‐ withPAM 2-PAM (to 200 μ (toM) 200 at 25M) °C. at Aliquots 25 ◦C. Aliquots(30 μL) were (30 µ assayedL) were as assayed in Figure as in3 at Figure the times3 at theindicated times indicatedon the x‐axis. on the Assayx-axis. values Assay v for values the solutionv for thecontaining solution 2 containing‐PAM (blue 2-PAM points that (blue correspond points that to correspond enzyme denaturation) to enzyme denaturation)were first fit to were Equation first fit(2) 1 5 towith Equation taged set (2) to 0 with and tfixedaged set initial to 0 estimates and fixed (min initial−1 for estimates k values) (min of kA− = for4 × 10k values)−5, kH = 4 of × 10kA−5,= kr4 = 2 10× 10− −,2 5 2 5 × k k E −5 k E andH = E40 = 100.01− Δ, A/minr = 2 to10 obtain− and kX 0= =2.20.01 × 10∆A and/min E totot obtain=0.69 ΔA/min.X = 2.2 These10− valuesand oftot t=aged0.69, kA∆, andA/min. Etot × × 5 × These values of t , k −5, and Etot along with k = 4 10 and kr = 0 were then fixed into Equation (2) along with kX = aged4 × 10A and kr = 0 were thenX fixed× into− Equation (2) to fit the assay values v for the tosolution fit the assay without values 2‐PAMv for (green the solution points without that reflect 2-PAM enzyme (green denaturation points that reflect and diethylphosphorylated enzyme denaturation and diethylphosphorylated enzyme hydrolysis). This fitting gave E = 0.003 ∆A/min and a k value of enzyme hydrolysis). This fitting gave E0 = 0.003 ΔA/min and a kH value0 of (47 + 3) × 10−6 minH−1 included (47 + 3) 10 6 min 1 included in Table1. in Table× 1. − −

2.4. 2-PAM Reactivation Rate Constants kR for Organophosphorylated AChE 2.4. 2‐PAM Reactivation Rate Constants kR for Organophosphorylated AChE The reactivation rate constants kr depend on the concentration of 2-PAM, but at the 2-PAM The reactivation rate constants kr depend on the concentration of 2‐PAM, but at the 2‐PAM concentrations employed here they were considerably larger than rate constants for denaturation, concentrations employed here they were considerably larger than rate constants for denaturation, aging and hydrolysis described above. Nevertheless, the determination of accurate solvent D2O aging and hydrolysis described above. Nevertheless, the determination of accurate solvent D2O isotope eﬀects required inclusion of these smaller rate constants in Equation (2). The protocol for isotope effects required inclusion of these smaller rate constants in Equation (2). The protocol for obtaining values of kr is shown in Figure5A. A rapid increase in assay values v was observed on obtaining values of kr is shown in Figure 5A. A rapid increase in assay values v was observed on adding organophosphorylated AChE to 2-PAM, followed by a much slower decrease in v that we adding organophosphorylated AChE to 2‐PAM, followed by a much slower decrease in v that we attributed to enzyme denaturation. The much longer slow decrease dominated the fitting procedure

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Molecules 2020, 25, x FOR PEER REVIEW 7 of 14 attributed to enzyme denaturation. The much longer slow decrease dominated the fitting procedure whenwhen thethe entireentire time time course course waswas analyzed,analyzed, so so fittingfitting was was divided divided into into two two steps. steps. InIn thethe firstfirst stepstep onlyonly the denaturation rate constant k was retained, and it showed a clear decrease in value at higher 2-PAM the denaturation rate constantX kX was retained, and it showed a clear decrease in value at higher 2‐ concentrations (Figure5A) as noted above. The second fitting step retained the value of k from the PAM concentrations (Figure 5A) as noted above. The second fitting step retained the valueX of kX from first step as an additional fixed variable, and k was fitted from the initial increase in v (Figure5A). the first step as an additional fixed variable, andr kr was fitted from the initial increase in v (Figure Values of k obtained at different 2-PAM concentrations were then analyzed according to Equation (4) 5A). Valuesr of kr obtained at different 2‐PAM concentrations were then analyzed according to to obtain the maximum reactivation rate constant k at concentrations of 2-PAM that saturated the Equation (4) to obtain the maximum reactivation rateR constant kR at concentrations of 2‐PAM that organophosphorylatedsaturated the organophosphorylated enzyme (Figure enzyme5B). (Figure 5B).

Figure 5. 2-PAM reactivation of dimethylphosphorylated AChE in H O. A mixture of 1.6 µM paraoxon Figure 5. 2‐PAM reactivation of dimethylphosphorylated AChE2 in H2O. A mixture of 1.6 μM methyl and 1.5 µM AChE in 100 mM sodium phosphate and 0.1% BSA (buffer) in H O (pH 8.0) was paraoxon methyl and 1.5 μM AChE in 100 mM sodium phosphate and 0.1% BSA (buffer)2 in H2O (pH incubated for 30 min at 25 C. A 2.5-fold excess of buffer in H O was added, and 100 µL was applied to 8.0) was incubated for 30 min◦ at 25 °C. A 2.5‐fold excess of buffer2 in H2O was added, and 100 μL was a 0.5 mL spin column that had been prewashed four times in buffer in H O and centrifuged at 1000 g applied to a 0.5 mL spin column that had been prewashed four times in 2buffer in H2O and centrifuged× for 1 min. The eluent was added to 215 µL of buffer in H O and cooled to 4 C, and 100 µL aliquots at 1000× g for 1 min. The eluent was added to 215 μL of buffer2 in H2O and cooled◦ to 4 °C, and 100 μL were added at time zero to 700 µL of buffer in H O containing final concentrations of 905, 185, or 24 µM aliquots were added at time zero to 700 μL of2 buffer in H2O containing final concentrations of 905, 2-PAM at 25 C. (A) Aliquots (30 µL) were assayed as indicated in Figure3 at the times indicated on 185, or 24 μM◦ 2‐PAM at 25 °C. (A) Aliquots (30 μL) were assayed as indicated in Figure 3 at the times the x-axis. Assay values v for the solutions containing 905 µM (green points), 185 µM (blue points) indicated on the x‐axis. Assay values v for the solutions containing 905 μM (green points), 185 μM or 24 µM 2-PAM (red points) were analyzed with Equation (2) in two steps, with taged set to 0 and (blue points) or 24 μM 2‐PAM (red points) were analyzed with Equation (2) in two steps, with taged set 3 1 3 1 kA and kH fixed at 2 10− min− and 6 10− min− , respectively, and kr, kX, Etot, and E0 as fitted to 0 and kA and kH fixed at 2 × 10−3 min−1 and 6 × 10−3 min−1, respectively, and kr, kX, Etot, and E0 as fitted × × 1 variables. In the first step, points over the entire 8800-min time course were fitted to obtain kX (in min− ) variables. In the first step, points over the entire 8800‐min time course were fitted to obtain kX (in 5 5 5 = 2.5 10− (green points), 6.3 10− (blue points), and 7.0 10− (red points). These kX were then min−1×) = 2.5 × 10−5 (green points),× 6.3 × 10−5 (blue points), and 7.0× × 10−5 (red points). These kX were then fixed along with kA and kH in the second step in which time courses of 250 to 450 min were fitted to fixed along with kA and kH in the second step in which time courses of 250 to 450 min were fitted to give more precise values of kr.(B) Values of kr from the second fitting step in panel A were then fitted r r give more precise values of k . (B) Values of k from the2 second1 fitting step in panel A were then fitted to Equation (4) to obtain values of kR (12.5 + 0.2) 10− min− included in Table1. The corresponding to Equation (4) to obtain values of kR (12.5 + 0.2)× × 10−2 min−1 included in Table 1. The corresponding fit of kr values obtained in D2O is also shown. Fitted values of KP were 170 µM in H2O and 90 µM in fit of kr values obtained in D2O is also shown. Fitted values of KP were 170 μM in H2O and 90 M in D2O. Error values typical of kr for both H2O and D2O data sets are shown with the D2O data points. D2O. Error values typical of kr for both H2O and D2O data sets are shown with the D2O data points. 2.5. Accuracy of Rate Constants for Reactions of Organophosphorylated hAChE 2.5. Accuracy of Rate Constants for Reactions of Organophosphorylated hAChE In addition to the rate constants given in Table1, we also determined kX values in the absence of In addition to the rate constants6 1 given in Table 1, we also determined6 kX values1 in the absence of 2-PAM in H2O ((53 7) 10− min− , n = 7) and in D2O (41 14) 10− min− , n = 3). These values ± × −6 −1 ± × −6 −1 are2‐PAM significant in H2O because, ((53 ± 7) in× 10 some min cases,, n several= 7) and measured in D2O (41 rate ± 14) constants × 10 min are close, n = 3). in value,These renderingvalues are significant because, in some cases, several measured rate constants are close in value, rendering the the accuracy of one rate constant highly dependent upon another. For example, values of kH, kA and accuracy of one rate constant highly dependent upon another. For example, values of kH, kA and kX kX for diethylphosphorylated AChE are almost identical in D2O. The rate constants most sensitive to for diethylphosphorylated AChE are almost identical in D2O. The rate constants most sensitive to these equivalencies are the kA values in Table1. These values were obtained by fitting Equation (3), these equivalencies are the kA values in Table 1. These values were obtained 6by fitting1 Equation (3), where the exponential rate constant is kA + kX. Therefore, from the 40 10− min− value of kA in where the exponential rate constant is kA + kX. Therefore, from the 40 × 10×−6 min−1 value of kA in Table 1, a 2‐fold increase in kX renders kA close to zero. The value of kX also plays an important, although lesser, role in the determination of kH for diethylphosphorylated AChE in D2O (Table 1 and Figure 4): a 2‐fold increase in the fixed value of kX in Equation (2) increases kH by 45%, whereas a 2‐fold decrease

Molecules 2020, 25, 4412 8 of 14

Table1, a 2-fold increase in kX renders kA close to zero. The value of kX also plays an important, although lesser, role in the determination of kH for diethylphosphorylated AChE in D2O (Table1 and Figure4): a 2-fold increase in the fixed value of kX in Equation (2) increases kH by 45%, whereas a 2-fold decrease in kX decreases kH by 20%. Values of kH, kA, and kX for dimethylphosphorylated AChE diverge to a much greater extent (see Table1), making their accuracy largely independent of each other. In view of these differences in the rate constant interdependence of kA and kH, the similarities in the solvent D2O isotope effects for dimethyl- and diethylphosphorylated AChE in Table1 are reassuring but possibly serendipitous.

Table 1. Rate constants for reactions of organophosphorylated AChE and their solvent D2O isotope eﬀects.

6 1 a 6 1 b Phosphoryl Substituents Reaction k (10− min− )H2O k (10− min− )D2O n k H2O/k DO Dimethyl- 1500 500 1400 500 2 1.1 0.1 Aging (kA) ± ± ± Diethyl- 39 7 c 41 4 c 1 1.0 0.2 ± ± ± Dimethyl- 6000 530 3100 360 2 1.9 0.1 Hydrolysis (kH) ± ± ± Diethyl- 83 12 41 5 3 2.0 0.1 ± ± ± Dimethyl- 120,000 6000 61,000 4000 2 1.96 0.04 2-PAM reactivation (k ) ± ± ± Diethyl-R 23,000 1500 15,000 300 2 1.68 0.14 ± ± ± a Values listed for rate constants k in H2O are means of n experiments with the standard error of the mean. Since each entry in this table included paired measurements in H2O and D2O, n values were always the same for both solvents. b The solvent D2O isotope effect was the ratio of a rate constant in H2O to that in D2O. It was the unweighted average of the isotope effects from the n experiments calculated individually for paired H2O and D2O reactions in each experiment. When n = 1, the standard error was determined from the combined errors of the H2O and D2O fits to Equation (3). c When n = 1, the standard error was determined from the error of the fit to Equation (3). However, the overall errors in kA for diethylphosphorylated AChE depend much more on the values of kX and kH than on replicate values of kA (see Section 2.5), and therefore replicate values of kA for these species were not made.

2.6. Solvent Deuterium Oxide Isotope Eﬀects on Reaction Rate Constants

Analyses of isotope eﬀects in D2O relative to H2O have been useful in clarifying details of the AChE reaction pathway with a variety of substrates [15,16], but we’ve found few reports of these isotope eﬀects on the reactions of phosphorylated AChE. In our experiments in this report, we paired rate constant determinations in H2O and D2O by running them on the same day, starting with the same stock AChE frozen aliquot and employing the same dilution sequence. Our results are presented in Table1, and they are examined in detail in the Discussion.

3. Discussion

3.1. Kinetics of Decarbamoylation and of the Hydrolysis of Organophosphorylated AChE Rate constants for organophosphorylated AChE in Table1 are in reasonable agreement with corresponding values previously reported in slightly different solvents, pH conditions and temperatures. Values of k = 3000 10 6 min 1, of k = 17,000 10 6 min 1, and of k = 480,000 10 6 min 1 were A × − − H × − − R × − − obtained for dimethylphosphorylated human AChE [17]. For diethylphosphorylated AChE, values of k = 170 10 6 min 1 and of k = 120 10 6 min 1 were obtained with the mouse enzyme [18] and of A × − − H × − − k = 60,000 10 6 min 1 for the human enzyme [19]. R × − − An increase in size of the carbamoyl or organophosphoryl group attached to AChE significantly reduced the value of k3 or kH for deacylation. In Table1, the kH for diethylphosphorylated AChE is about 75-fold lower than that for dimethylphosphorylated AChE. A similar trend was seen with carbamoylated AChE, where k3 for N,N-diethylcarbamoyl AChE was about 300-fold lower than that for N,N-dimethylcarbamoyl AChE [2]. The difference for the organophosphorylated AChEs may result from distortion of the AChE active site by the larger diethylphosphoryl group as seen in Figure2A. The organophosphoryl group size appears less important for oxime reactivation, as kR in Table1 for 2-PAM reactivation of diethylphosphorylated AChE is only 5-fold lower than that for Molecules 2020, 25, 4412 9 of 14

Moleculesfor 2-PAM2020, 25reactivation, 4412 of diethylphosphorylated AChE is only 5-fold lower than that9 offor 14 dimethylphosphorylated AChE. To address the consequences of active site distortion more directly, we analyzed the solvent D2O isotope effects in the following section. dimethylphosphorylated AChE. To address the consequences of active site distortion more directly, we analyzed the solvent D O isotope effects in the following section. 3.2. Solvent Deuterium Oxide2 Isotope Effects on Rate Constants for Reactions of Organophosphorylated 3.2.AChE Solvent Deuterium Oxide Isotope Effects on Rate Constants for Reactions of Organophosphorylated AChE When proton transfer occurs in the rate-limitingrate-limiting step of a reaction,reaction, the raterate constantconstant for thatthat reaction shows a solvent D2O isotope effect. Rate-limiting proton transfer in an acylation or reaction shows a solvent D2O isotope effect. Rate-limiting proton transfer in an acylation or deacylation deacylation reaction results in a rate constant decrease of 2 to 3-fold when D2O replaces H2O as the reaction results in a rate constant decrease of 2 to 3-fold when D2O replaces H2O as the solvent [20]. solvent [20]. AChE-catalyzed acetylcholine hydrolysis falls well within this range, as kcat (a AChE-catalyzed acetylcholine hydrolysis falls well within this range, as kcat (a combination of acylation combination of acylation and deacylation rate constants) has a solvent D2O isotope effect of 2.4 and deacylation rate constants) has a solvent D2O isotope effect of 2.4 [15,21]. However, the isotope effect [15,21]. However, the isotope effect drops from 2.4 for kcat to 1.1–1.2 for the second order hydrolysis drops from 2.4 for kcat to 1.1–1.2 for the second order hydrolysis rate constant kcat/Kapp (also denoted rate constant kcat/Kapp (also denoted kE) with good substrates of AChE like acetylcholine. To interpret kE) with good substrates of AChE like acetylcholine. To interpret this drop, we proposed that proton this drop, we proposed that proton transfer in the acylation step k2 was rate-limiting for kcat but that transfer in the acylation step k2 was rate-limiting for kcat but that an earlier step like substrate binding, oran anearlier induced-fit step like conformational substrate binding, change or that an induced-fit does not involve conformational proton transfer, change was that rate-limiting does not involve for the proton transfer, was rate-limiting for the second order rate constant kcat/Kapp [15]. More explicitly, the second order rate constant kcat/Kapp [15]. More explicitly, the solvent D2O isotope effect is determined bysolvent the commitment D2O isotope toeffect catalysis, is determined denoted Cby [16 the]. Incommitme the verynt simple to catalysis, case of denoted Scheme3 Cand [16]. Equation In the very (1), simple case of Scheme 3 and Equation (1), acetylcholine (S) binds to enzyme E with an association acetylcholine (S) binds to enzyme E with an association rate constant of kS and a dissociation rate rate constant of kS and a dissociation rate constant of k-S to give an ES complex. constant of k-S to give an ES complex.

Scheme 3. Pathway for hydrolysis of acetylthiocholine (S) by AChE (E). Scheme 3. Pathway for hydrolysis of acetylthiocholine (S) by AChE (E).

Subsequent acylation with a rate constant k2 gives an acyl enzyme and choline products, here just Subsequent acylation with a rate constant k2 gives an acyl enzyme and choline products, here denoted P. The observed rate constant kE is given by Equation (1). The acylation step k2 involves proton just denoted P. The observed rate constant kE is given by Equation (1). The acylation step k2 involves transfer and has an isotope effect of 2–3, while kS and k S are assumed not to involve proton transfer. proton transfer and has an isotope effect of 2–3, while− kS and k−S are assumed not to involve proton The commitment to catalysis is defined as C = k2/k S. When C is small, kE = kS k2/k S, and kE shows an transfer. The commitment to catalysis is defined− as C = k2/k−S. When C is small, −kE = kS k2/k−S, and kE isotope effect. When C is large, kE = kS and it does not. shows an isotope effect. When C is large, kE = kS and it does not. k k k = 𝑘S 2𝑘 (1) E ( + ) 𝑘 = k S k2 (1) (𝑘− +𝑘) A comparable interpretation may be applied to the aging reaction in Scheme2. This reaction is enzymeA comparable catalyzed [22 interpretation] and involves may loss be of anapplied alkyl groupto the asaging the organophosphorylreaction in Scheme adduct 2. This is reaction converted is fromenzyme a phosphate catalyzed triester [22] and to ainvolves diester. Whileloss of a protonan alkyl is transferredgroup as the to theorganophosphoryl alkyl leaving group adduct in this is converted from a phosphate triester to a diester. While a proton is transferred to the alkyl leaving reaction, this transfer does not appear to be rate limiting as the solvent D2O isotope effects for the group in this reaction, this transfer does not appear to be rate limiting as the solvent D2O isotope aging reaction rate constant kA in Table1 are about 1.0. We have found no other reports of isotope eeffectsffects onfor agingthe aging of dimethyl- reaction rate or diethylphosphorylated constant kA in Table 1 are AChE, about but 1.0. a We similar have isotope found no eff ectother of reports 1.2 has beenof isotope reported effects for on aging agin ofg 2-propoxy-methylphosphonylatedof dimethyl- or diethylphosphorylat AChEed AChE, [23]. but a similar isotope effect of 1.2We has next been asked reported whether for aging the hydrolysisof 2-propoxy-methylphosphon and oxime reactivationylated reactions AChE [23]. in Scheme 2 involve generalWe acid-basenext asked catalysis whether with the rate-limiting hydrolysis and proton oxim transfer.e reactivation To facilitate reactions the analysis,in Scheme we 2 consider involve Schemegeneral4 acid-base, which shows catalysis a slight with extension rate-limiting of Scheme proton1 in transfer. which a To second facilitate acyl enzymethe analysis, species we is consider added inScheme conformational 4, which shows equilibrium a slight extension with the first.of Scheme In particular, 1 in which Scheme a second4 includes acyl enzyme an inactive species enzymeis added in conformational equilibrium with the first. In particular, Scheme 4 includes an inactive enzyme form (E1C or E1OP) with a distorted active site and an active enzyme form (E2C or E2OP) that can form (E1C or E1OP) with a distorted active site and an active enzyme form (E2C or E2OP) that can undergo hydrolysis with a rate constant k3 or kH. The forward and reverse rate constants for these undergo hydrolysis with a rate constant k3 or kH. The forward and reverse rate constants for these equilibria are kF and k-F, respectively, and they are assumed not to involve proton transfer. The general equilibria are kF and k-F, respectively, and they are assumed not to involve proton transfer. The general solution to Scheme4 is more complicated than that of Scheme3, as neither E2C nor E2OP necessarily issolution in the steadyto Scheme state. 4 is However, more complicate two extremed than cases that mayof Scheme be considered 3, as neither in which E2C nor these E2OP intermediates necessarily is in the steady state. However, two extreme cases may be considered in which these intermediates are in the steady state: (1) the commitment to catalysis k3/k F or kH/k F is small, and the deacylation are in the steady state: (1) the commitment to catalysis k3/k−F− or kH/k−F is− small, and the deacylation rate rate constant k3kF/(k F+k3) or kHkF/(k F+kH) shows the solvent D2O isotope effect inherent in k3 or kH; constant k3kF/(k−F+k3)− or kHkF/(k−F+kH) shows− the solvent D2O isotope effect inherent in k3 or kH; (2) the (2) the commitment to catalysis k3/k F or kH/k-F is large, the acyl intermediates are not equilibrated and −

Molecules 2020, 25, x FOR PEER REVIEW 10 of 14 Molecules 2020, 25, 4412 10 of 14 Molecules 2020, 25, 4412 10 of 14 commitment to catalysis k3/k−F or kH/k‐F is large, the acyl intermediates are not equilibrated and the commitment to catalysis k3/k−F or kH/k-F is large, the acyl intermediates are not equilibrated and the deacylation rate constant k3kF/(k−F+kH) or kHkF/(k−F+kH) shows only a fraction of the isotope effect thedeacylation deacylation rate rate constant constant k3k F/(k k/−(Fk+kH+) kor) orkHkkF/(kk−F/(+kkH) +showsk ) shows only only a fraction a fraction of ofthe the isotope isotope effect eﬀect inherent in k3 or kH and no isotope3 FeffectF if Hk3 or kHH >>F k−F. F H inherent in k3 or kH and no isotope effect− if k3 or kH >>− k−F. inherent in k3 or kH and no isotope eﬀect if k3 or kH >> k F. −

SchemeScheme 4. Extension 4. Extension of Scheme of Scheme 1 to1 toinclude include an an inactive inactive enzyme enzyme form form (E (1CE 1orC E or1OP).E1OP). Scheme 4. Extension of Scheme 1 to include an inactive enzyme form (E1C or E1OP).

WeWe recently recently investigated investigated the the hydrolysis hydrolysis of ofcarbamoylated carbamoylated AChEs AChEs for for a series a series of carbamate of carbamate esters esters We recently investigated the hydrolysis of carbamoylated AChEs for a series of carbamate esters andand obtained obtained the the solvent solvent D2O D 2isotopeO isotope effects effects summarized summarized in inFigure Figure 6 [2].6[ 2 The]. The rate rate constant constant k3 fork3 for the the and obtained the solvent D2O isotope effects summarized in Figure 63 [2]. The1 rate constant k3 for the smallestsmallest carbamoyl carbamoyl group, group, an an N‐Nmonomethylcarbamoyl,-monomethylcarbamoyl, was was 12 12 × 1010−3 −minmin−1, −and, and its isotope its isotope effect effect of of smallest carbamoyl group, an N-monomethylcarbamoyl, was 12× × 10−3 min−1, and its isotope effect of 2.82.8 was was consistent consistent with with rate rate-limiting‐limiting proton proton transfer transfer and and a small a small value value of C. of However, C. However, as asthe the N‐alkylN-alkyl 2.8 was consistent with rate-limiting proton transfer and a small value of C. However, as the N-alkyl groupsgroups on on carbamoylated carbamoylated AChEs AChEs increased increased in in size, size, the the decarbamoylation decarbamoylation rate rate constant constant k33 decreaseddecreased to groups on carbamoylated3 1 AChEs increased in size, the decarbamoylation rate constant k3 decreased to aboutabout 0.02 0.02 × 1010−3− minmin−1 −forfor N,NN‐,diethylcarbamoylatedN-diethylcarbamoylated AChE AChE and and the the isotope isotope effect effect was was only only slightly slightly to about 0.02× × 10−3 min−1 for N,N-diethylcarbamoylated AChE and the isotope effect was only slightly aboveabove 1 (Figure 1 (Figure 6),6 indicating), indicating a high a high value value of ofC. C.In Inthat that report report we we noted noted the the crystallographic crystallographic evidence evidence above 1 (Figure 6), indicating a high value of C. In that report we noted the crystallographic evidence of activeof active site site distortion distortion in diethylphosphorylated in diethylphosphorylated AChE AChE shown shown in Figure in Figure 2A,2A, and and we we suggested suggested that that of active site distortion in diethylphosphorylated AChE shown in Figure 2A, and we suggested that a largera larger size size of ofthe the carbamoyl carbamoyl group group is likely is likely to tobe bean animportant important factor factor in a in shift a shift away away from from proton proton a larger size of the carbamoyl group is likely to be an important factor in a shift away from proton transfertransfer in the in the rate rate-limiting‐limiting step step for fork3. Itk 3may. It may be useful be useful to explore to explore molecular molecular modeling modeling of the of array the array of transfer in the rate-limiting step for k3. It may be useful to explore molecular modeling of the array of conformationalof conformational variants variants available available in AChEs in AChEs with large with carbamoyl large carbamoyl groups groups to obtain to obtainsupport support for this for conformational variants available in AChEs with large carbamoyl groups to obtain support for this proposal.this proposal. proposal.

Figure 6. k Solvent D2O isotope effects on the AChE decarbamoylation rate constant 3 at 25 ◦C[2]. Figure 6. Solvent D2O isotope effects on the AChE decarbamoylation rate constant k3 at 25 °C [2]. WeFigure designed 6. Solvent the experiments D2O isotope here effects to examineon the AChE whether decarbamoylation active site distortion rate constant in diethylphosphorylated k3 at 25 °C [2]. AChEWe indesigned fact does resultthe experiments in a shift away here from rate-limitingto examine proton whether transfer active in the site hydrolysis distortion and oximein We designed the experiments here to examine whether active site distortion in diethylphosphorylatedreactivation reactions AChE of this in AChEfact does intermediate. result in a shift For away comparison, from rate we‐limiting included proton measurements transfer in of diethylphosphorylated AChE in fact does result in a shift away from rate-limiting proton transfer in thesolvent hydrolysis D2O and isotope oxime effects reactivation for dimethylphosphorylated reactions of this AChE AChE, intermediate. an intermediate For unlikelycomparison, to involve we activethe hydrolysis site distortion and oxime [10,12 ].reactivation Oxime reactivation, reactions of like this hydrolysis AChE intermediate. in Scheme2, involvesFor comparison, attack of we a included measurements of solvent D2O isotope effects for dimethylphosphorylated AChE, an included measurements of solvent D2O isotope effects for dimethylphosphorylated AChE, an intermediatenucleophile unlikely on the to tetravalent involve active phosphorus site distortion to presumably [10,12]. Oxime form a reactivation, pentavalent like transition hydrolysis state in [24 ]. intermediate unlikely to involve active site distortion [10,12]. Oxime reactivation, like hydrolysis in SchemeThis state 2, involves then decomposes attack of a to nucleophile regenerate activeon the enzyme tetravalent and anphosphorus organophosphorylated to presumably oxime form [7 ,a25 ], Scheme 2, involves attack of a nucleophile on the tetravalent phosphorus to presumably form a pentavalentand both transition the formation state and [24]. decomposition This state then of decomposes this state are to likely regenerate to involve active rate-limiting enzyme and proton an pentavalent transition state [24]. This state then decomposes to regenerate active enzyme and an organophosphorylatedtransfer. In the structure oxime in [7,25], Figure and2B, 50 both mM the 2-PAM formation was and di ff useddecomposition into the crystal of this [ 9state]. This are structure likely organophosphorylated oxime [7,25], and both the formation and decomposition of this state are likely to suggestsinvolve rate that‐limiting 2-PAM stabilizesproton transfer. the active In theE2OP structure species in in Figure Scheme 2B,4, perhaps50 mM 2 to‐PAM the exclusion was diffused of the distortedto involveE rate-limitingOP species, andprotonk fortransfer. diethylphosphorylated In the structure in AChE Figure in 2B, Table 501 mMis only 2-PAM 5-fold was lower diffused than into the crystal1 [9]. This structureR suggests that 2‐PAM stabilizes the active E2OP species in Scheme thatinto forthe dimethylphosphorylatedcrystal [9]. This structure AChE.suggests These that observations2-PAM stabilizes suggest the thatactive the E concentration2OP species in ofSchemeE OP 4, perhaps to the exclusion of the distorted E1OP species, and kR for diethylphosphorylated AChE in1 4, perhaps to the exclusion of the distorted E1OP species, and kR for diethylphosphorylated AChE in Tablebound 1 is only to 2-PAM 5‐fold is lower negligible than that and for that dimethylphosphorylatedkR will reflect the rate-limiting AChE. protonThese observations transfer inherent suggest when Table 1 is only 5-fold lower than that for dimethylphosphorylated AChE. These observations suggest that the concentration of E1OP bound to 2‐PAM is negligible and that kR will reflect the rate‐limiting that the concentration of E1OP bound to 2-PAM is negligible and that kR will reflect the rate-limiting

Molecules 2020, 25, 4412 11 of 14

both acyl groups migrate to the oxime. The solvent D2O isotope effects of 2.0 and 1.7 for the dimethyl- and diethylphosphorylated AChE are consistent with this interpretation. Finally, we examined the rate constants kH for hydrolysis of both diethylphosphorylated AChE and dimethylphosphorylated AChE to determine if their solvent D2O isotope effects reflect the fact that only diethylphosphorylated AChE appears to form the distorted active conformation in Figure2A. The values in Table1 do not support this proposal, as both organophosphorylated species show isotope effects that are close to 2.0. This value, although somewhat low for typical enzyme deacylation, suggests that any distorted conformation E1OP involving diethylphosphorylated AChE equilibrates relatively rapidly with the active conformation E2OP and that the commitment to catalysis C = kH/k F − is small. These isotope effects for organophosphorylated AChE hydrolysis are significant because this reaction, of those in Scheme2, bears the closest resemblance to the hydrolysis of the carbamoylated enzymes in Figure6. Furthermore, the conclusion above that the commitment to catalysis C = k3/k F is − large for N,N-diethylcarbamoylated AChE while C = kH/k F is small for diethylphosphorylated AChE − is difficult to justify. Their respective rate constants, k = 0.02 10 3 min 1 for decarbamoylation and 3 × − − k = 0.08 10 3 min 1 for dephosphorylation (Table1 and Figure6), are too similar to support these H × − − opposing conclusions about C if the same value of k-F holds for both acylated AChEs.

4. Materials and Methods

4.1. Reagents Recombinant hAChE was expressed as a secreted, disulfide-linked dimer in drosophila S2 cells and purified by affinity chromatography as outlined previously [26]. Initial 0.5 mL fractions were maintained in 5 mM decamethonium bromide (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) at 4 ◦C. Fractions were dialyzed against 20 mM sodium phosphate and 0.02% Triton X-100 (pH 7.0) at 4 C, and 10- or 20- µL aliquots of the dialyzates were frozen at 20 C until use. O,O-Diethyl ◦ − ◦ O-(4-nitrophenyl) phosphate (paraoxon) and O,O-dimethyl O-(4-nitrophenyl) phosphate (paraoxon methyl) were commercial samples from Sigma-Aldrich Chemical Co (St. Louis, MO, USA).

4.2. Assay of Substrate Hydrolysis Hydrolysis rates v for the substrate acetylthiocholine were measured in a coupled Ellman reaction in which thiocholine generated in the presence of the indicated concentration of DTNB was determined by 1 1 the formation of the thiolate dianion of DTNB at 412 nm (∆ε412 nm = 14,150 M− cm− )[27]. Total AChE concentrations (Etot) could be calculated assuming 450 units/nmol [28]. One unit of AChE activity corresponds to 1 µmol of acetylthiocholine hydrolyzed/min under standard pH-stat assay conditions at pH 8 [28,29]. Our conventional spectrophotometric assay at 412 nm was conducted in pH 7.0 buffer. With wild type hAChE and 0.5 mM acetylthiocholine, this assay resulted in 4.8 ∆A412 nm/min with 1 nM AChE or about 76% of the pH stat assay standard, but here Etot was expressed simply in terms of ∆A412/min. The assay mixture here contained final concentrations of acetylthiocholine and DTNB of 1.0 mM and 0.3 mM, respectively, in 100 mM sodium phosphate and 0.1% BSA at pH 8.0. Aliquots of AChE were added to a final volume was 3.0 mL, and the assay was conducted at 25 ◦C. Absorbance at 412 nm was recorded with time on a Varian Cary 3A spectrophotometer.

4.3. Reactions of Organophosphorylated hAChE We generated organophosphorylated AChE (EOP in Scheme1) from either paraoxon (to give diethylphosphorylated AChE) or paraoxon methyl (to give dimethylphosphorylated AChE) as outlined in the legends to Figures3–5. Experimental traces of organophosphorylated AChE reactions measured with acetylthiocholine activity assays (v) were obtained and the equation for the general solution used to ﬁt these traces to Scheme2 is given in Equation (2). Data were ﬁtted to Equation (2) by unweighted Molecules 2020, 25, 4412 12 of 14

nonlinear regression with SigmaPlot (version 12.0, Systat Software Inc., Chicago, IL, USA). When kr >> 1 kH + kA + kX and t = taged >> kr− , Equation (2) reduces to Equation (3). k t (kr+k +k +k )t (kr + kH) e− X e− H A X kAtaged kXt v = Etote− E0 − + E0 e− (2) − (kr + kH + kA)

(kA+kX)taged v = Etote− (3)

In Equations (2) and (3), taged is the time that enzyme and OP were incubated prior to the start of the measured reaction, t is the duration in min of the measured reaction and E0 is the initial enzyme concentration (∆A412/min at t = 0). Values of the second order reactivation rate constants kr were obtained at various 2-PAM concentrations that were run in parallel, and these values were analyzed with Equation (4) to obtain the maximal ﬁrst order reactivation rate constant kR at saturating concentrations of 2-PAM and the dissociation constant KP for 2-PAM binding to organophosphorylated AChE.

kR kr = (4) + KP 1 [2 PAM] − 4.4. Solvent Deuterium Oxide Isotope Effects Reactions of organophosphorylated AChE were conducted in 100 mM sodium phosphate and 0.1% BSA at pH 8.0. The pH was the uncorrected value read by the pH meter for both H2O and D2O buffers, and the pH of 8.0 (rather than more typical pH values of 7.0–7.4 for AChE studies) was selected to minimize enzyme pKa differences [3] between H2O and D2O. Organophosphorylated AChE reactions in D2O were paired with reactions in H2O to maximize precision in comparing rates. While frozen stocks of AChE were in H2O, all subsequent dilutions were identical in either H2O or D2O. The percentage H2O in an assayed D2O aging reaction mixture was 3%, and the maximum percentage in hydrolysis or oxime reactivation mixtures was 1%.

Funding: This research received no external funding. Conﬂicts of Interest: The author declares no competing ﬁnancial interests.

Abbreviations AChE: acetylcholinesterase; BSA, bovine serum albumin; hAChE, human acetylcholinesterase; mAChE, mouse acetylcholinesterase; TcAChE, Torpedo californica acetylcholinesterase; DTNB, 5,5’-dithiobis-(2-nitrobenzoic acid); 2-PAM, 2-pyridinealdoxime methiodide.

References

1. Rosenberry, T.L. Quantitative simulation of endplate currents at neuro-muscular junctions based on the reactions of acetylcholine with acetylcholine receptor and acetylcholinesterase. Biophys. J. 1979, 26, 263–290. [CrossRef] 2. Venkatasubban, K.S.; Johnson, J.L.; Thomas, J.L.; Fauq, A.; Cusack, B.; Rosenberry, T.L. Decarbamoylation of acetylcholinesterases is markedly slowed as carbamoyl groups increase in size. Arch. Biochem. Biophys. 2018, 655, 67–74. [CrossRef][PubMed] 3. Reiner, E.; Aldridge, W.N. Eﬀect of pH on inhibition and spontaneous reactivation of acetylcholinesterase treated with esters of phosphorus acids and of carbamic acids. Biochem. J. 1967, 105, 171–179. [CrossRef] [PubMed] 4. Fukuto, T.R. Mechanism of action of organophosphorus and carbamate insecticides. Environ. Health Perspect. 1990, 87, 245–254. [CrossRef] 5. Myers, D.K. Studies on cholinesterase. 10. Return of cholinesterase activity in the rat after inhibition by carbamoyl ﬂuorides. Biochem. J. 1956, 62, 556–563. Molecules 2020, 25, 4412 13 of 14

6. Froede, H.C.; Wilson, I.B. Acetylcholinesterase. In The Enzymes, 3rd ed.; Boyer, P.D., Ed.; Academic Press: New York, NY, USA, 1971; pp. 87–114. 7. Wilson, I.B. Acetylcholinesterase. XI. Reversibility of tetraethyl pyrophosphate inhibiton. J. Biol. Chem. 1951, 190, 111–117. 8. Rosenberry, T.L.; Cheung, J. Rate-limiting step in the decarbamoylation of acetylcholinesterases with large carbamoyl groups. Chem. Biol. Interact. 2019, 308, 392–395. [CrossRef] 9. Franklin, M.C.; Rudolph, M.J.; Ginter, C.; Cassidy, M.S.; Cheung, J. Structures of paraoxon-inhibited human acetylcholinesterase reveal perturbations of the acyl loop and the dimer interface. Proteins 2016, 84, 1246–1256. [CrossRef] 10. Hörnberg, A.; Tunemalm, A.K.; Ekström, F. Crystal structures of acetylcholinesterase in complex with organophosphorus compounds suggest that the acyl pocket modulates the aging reaction by precluding the formation of the trigonal bipyramidal transition state. Biochemistry 2007, 46, 4815–4825. [CrossRef] 11. Millard, C.B.; Kryger, G.; Ordentlich, A.; Greenblatt, H.M.; Harel, M.; Raves, M.L.; Segall, Y.; Barak, D.; Shafferman, A.; Silman, I.; et al. Crystal structures of aged phosphonylated acetylcholinesterase: Nerve agent reaction products at the atomic level. Biochemistry 1999, 38, 7032–7039. [CrossRef] 12. Millard, C.B.; Koellner, G.; Ordentlich, A.; Shafferman, A.; Silman, I.; Sussman, J.L. Reaction products of acetylcholinesterase and VX reveal a mobile histidine in the catalytic triad. J. Am. Chem. Soc. 1999, 121, 9883–9884. [CrossRef] 13. Kovalevsky, A.; Blumenthal, D.K.; Cheng, X.; Taylor, P.; Radic, Z. Limitations in current acetylcholinesterase structure–based design of oxime antidotes for organophosphate poisoning. Ann. N. Y. Acad. Sci. 2016, 1378, 41–49. [CrossRef][PubMed] 14. Michel, H.O.; Hackley, B.E.; Berkowitz, L.; List, G.; Hackley, E.B.; Gillilan, W.; Pankau, M. Ageing and dealkylation of soman (pinacolylmethylphosphonofluoridate)-Inactivated eel cholinesterase. Arch. Biochem. Biophys. 1967, 121, 29–34. [CrossRef] 15. Rosenberry, T.L. Catalysis by acetylcholinesterase. Evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis. Proc. Natl. Acad. Sci. USA 1975, 72, 3834–3838. 16. Quinn, D.M. Acetylcholinesterase: Enzyme structure, reaction dynamics, and virtual transition states. Chem. Rev. 1987, 87, 955–979. [CrossRef] 17. Worek, F.; Diepold, C.; Eyer, P.Dimethylphosphoryl-inhibited human cholinesterases: Inhibition, reactivation, and aging kinetics. Arch. Toxicol. 1999, 73, 7–14. [CrossRef] 18. Jennings, L.L.; Malecki, M.; Komives, E.A.; Taylor, P. Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry. Biochemistry 2003, 42, 11083–11091. [CrossRef] 19. Grosfeld, H.; Barak, D.; Ordentlich, A.; Velan, B.; Shafferman, A. Interactions of oxime reactivators with diethylphosphoryl adducts of human acetylcholinesterase and its mutant derivatives. Mol. Pharmacol. 1996, 50, 639–649. 20. Bender, M.L.; Hamilton, G.A. Kinetic isotope effects of deuterium oxide on several a-chymotrypsin-catalyzed reactions. J. Am. Chem. Soc. 1962, 84, 2570–2576. [CrossRef] 21. Bender, M.L.; Clement, G.E.; Kezdy, F.J.; Heck, H.D.A. The correlation of the pH (pD) dependence and the stepwise mechanism of α-chymotrypsin-catalyzed reactions. J. Am. Chem. Soc. 1964, 86, 3680–3690. [CrossRef] 22. Qian, N.; Kovach, I.M. Key active site residues in the inhibition of acetylcholinesterases by soman. FEBS Lett. 1993, 336, 263–266. [CrossRef] 23. Kovach, I.M.; Bennet, A.J. Comparative study of nucleophilic am) enzymic reactions of 2-propyl methylphosphonate derivatives. Phosphorus Sulfur Silicon Relat. Elem. 1990, 51, 51–56. [CrossRef] 24. Wong, L.; Radic, Z.; Bruggemann, R.J.M.; Hosea, N.; Berman, H.A.; Taylor, P.Mechanism of oxime reactivation of acetylcholinesterase analyzed by chirality and mutagenesis. Biochemistry 2000, 39, 5750–5757. [CrossRef] [PubMed] 25. Luo, C.; Saxena, A.; Smith, M.; Garcia, G.; Radic, Z.; Taylor, P.; Doctor, B.P. Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium. Biochemistry 1999, 38, 9937–9947. [CrossRef][PubMed] Molecules 2020, 25, 4412 14 of 14

26. Mallender, W.D.; Szegletes, T.; Rosenberry, T.L. Organophosphorylation of acetylcholinesterase in the presence of peripheral site ligands: Distinct effects of propidium and fasciculin. J. Biol. Chem. 1999, 274, 8491–8499. [CrossRef][PubMed] 27. Johnson, J.L.; Cusack, B.; Davies, M.P.; Fauq, A.; Rosenberry, T.L. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate. Biochemistry 2003, 42, 5438–5452. [PubMed] 28. De Ferrari, G.V.; Mallender, W.D.; Inestrosa, N.C.; Rosenberry, T.L. Thioflavin T is a fluorescent probe of the acetylcholinesterase peripheral site that reveals conformational interactions between the peripheral and acylation sites. J. Biol. Chem. 2001, 276, 23282–23287. [CrossRef] 29. Rosenberry, T.L.; Scoggin, D.M. Structure of human erythrocyte acetylcholinesterase. Characterization of intersubunit disulfide bonding and detergent interaction. J. Biol. Chem. 1984, 259, 5643–5652.

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