Ns

JI

a

AA

KASND

1

eibt(bfatrattgsDpe

0d

Chemico-Biological Interactions 187 (2010) 128–134

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

on-productive binding of butyryl(thio)choline in the activeite of vertebrate acetylcholinesterase

ure Stojan ∗

nstitute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov Trg 2, 1000 Ljubljana, Slovenia

r t i c l e i n f o

rticle history:vailable online 7 May 2010

eywords:cetylcholinesteraselow inhibitionon-productive binding

a b s t r a c t

The kinetic behavior of cholinesterases is unconventional. While their activities are higher than expectedby classical Michaelis–Menten reaction mechanisms, at intermediate substrate concentrations they showstrong inhibition by excess of substrate. To date, the main explanations used for all of their kineticpeculiarities include hindrance of product exit, entropically improved water orientation by a second sub-strate molecule, and complete blockade of the fully occupied active site. However, with the hydrolysisof butyryl(thio)choline by vertebrate acetylcholinesterase, there are time-dependent and substrate-

ifferential equationsconcentration-dependent decreases in catalytic activity. As the substrate depletion results in the expecteddownwardly concave shape of the progress curves for product formation at low substrate concentrations,this cannot be the reason for the bending of the linear progress curves at higher substrate concentrations.A good theoretical and practical explanation was reached by including the time-dependent appearanceof a non-productive enzyme–substrate complex in the reaction scheme. The slow establishment of thiscomplex appears to be a rare occurrence of incorrect substrate orientation at the bottom of the activesite, with this blocked by a second substrate molecule.

© 2010 Elsevier Ireland Ltd. All rights reserved.

. Introduction

Acetylcholinesterases (AChEs) are highly specific and veryffective enzymes. While their natural substrate, acetylcholine,s hydrolyzed very rapidly, the turnover number of the car-oxylic ester substrate decreases sharply for acyl groups largerhan propionyl [1]. This is why the rate of butyryl(thio)cholineBTCh) hydrolysis by vertebrate enzymes has been reported toe negligible, as seen with electric-eel AChE and Torpedo cali-

ornica AChE (TcAChE). It has been shown that the size of thecyl moiety of BTCh results in its greatly reduced accommoda-ion in the acyl pocket of the AChEs. This is seen by the increasedate of BTCh hydrolysis by a mutant AChE enzyme (by as muchs 40% of that for acetyl(thio)choline [ATCh]) following substi-ution of the two phenylalanines that line the acyl pocket byhe smaller aliphatic chains of leucine and valine [2]. This is inood agreement with the very similar rates of BTCh hydrolysis

een for wild-type insect AChE (from Drosophila melanogaster;mAChE), which lacks one phenylalanine at the correspondingosition, and the double mutant of a vertebrate AChE. How-ver, the situation is not so straightforward, since the DmAChE

∗ Tel.: +386 1 5437649; fax: +386 1 5437641.E-mail address: stojan@mf.uni-lj.si.

009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2010.05.001

mutant with two phenylalanines does not lose activity towardsBTCh.

It has been shown that apart from their hydrolysis of carboxylicesters, vertebrate AChEs can cleave between bonds, including thepeptide bond [3,4]. This occurs at very low rates, so that con-siderably high enzyme concentrations are needed to obtain ameasurable activity [5]. However, even with such substrates, theconcentrations of the enzyme in the kinetic measurements aremany magnitudes lower than those of the substrate, thus preserv-ing the basic assumption of classical enzyme kinetics.

The reactions of AChEs with poor substrates are particularlyinteresting from the mechanistic point of view. As the rate limit-ing steps differ, it is possible to use these kinetics to reveal reactionintermediates that do not accumulate in the reaction with the natu-ral substrate. Recently, a homotropic acceleration of acetylation bypara-nitro phenyl acetate was reported, in contrast to the partiallyblocked acetylation by ATCh. This was interpreted as improvedaccommodation of a rigid aromatic substrate at the bottom of theactive site upon simultaneous binding of a second molecule at theperipheral site [6]. Under the same conditions, a rapidly reacting

substrate would block the exit of its products, and in this waydecrease the rate of it own turnover [7].

All of these considerations led us to investigate in detail thehydrolysis of BTCh itself and the effects of BTCh on the hydrolysisof ATCh, by TcAChE.

l Inter

2

2

rtCw

2

iaacatImoT

2

tp(ytfABtiutcw2fL

2

tmyftfa

2T

cpwdtd

This approach allowed the determination of a unique and commonset of kinetic constants (Table 1). It should also be stressed that theseven kinetic constants for the hydrolysis of ATCh are well withinthe experimental error of previously published data for the samesystem [11]. On the other hand, there are five new constants for the

J. Stojan / Chemico-Biologica

. Materials and methods

.1. Chemicals

ATCh, BTCh and dithio-bis-di-nitro benzoic acid (DTNB; Ellman’seagent) were from Sigma (St Louis, MO, USA), and m-(N,N,N-rimethylammonio)-2,2,2-trifluoroacetophenone (TMTFA) fromalbiochem. All other reagents were of analytical grade. TcAChEas a gift from Prof. Israel Silman (Rehovot, Israel).

.2. Enzyme titration

AChE concentrations were determined by so-called pseudo-rreversible titration [8], using the transition state analog TMTFAs the titrating agent. The residual enzyme activity was measuredfter 5 min preincubation of each enzyme sample of unknown con-entration with increasing concentrations of the transition statenalog TMTFA. The curves obtained were analyzed according tohe equation for the irreversible second-order complex formation.n this way, the enzyme concentrations initially used were deter-

ined in three different experiments simultaneously. An activityf 0.64 units of optical density per minute was established for 1 nMcAChE and 0.5 mM ATCh.

.3. Substrate hydrolysis measurements

The progress curves for the time courses of product forma-ion for the hydrolysis of BTCh and of ATCh in the absence andresence of BTCh were measured on a stopped-flow apparatusHi-Tech, Salisbury, UK). The enzymatic and non-enzymatic hydrol-sis of ACTh was followed by Ellman’s method [9], by measuringhe absorbance change at 412 nm. These measurements were per-ormed at ATCh and BTCh concentrations from 5 �M to 75 mM;TCh hydrolysis was also measured in the presence of two differentTCh concentrations (0.5 mM and 5 mM). At low substrate concen-rations (5–75 �M), the hydrolysis was measured to completion;n all of the other measurements, the reactions were followedntil 60–80 �M product was formed. The concentration of DTNB athe beginning of each measurement was 0.66 mM, and each timeourse was carried out twice, except for the hydrolysis of BTCh,hich was followed five times. The experiments were carried out at

5 ◦C in 25 mM phosphate buffer, pH 7.0. For the experiments per-ormed using a conventional spectrophotometer, a Perkin-Elmerambda45 UV/Vis spectrometer was used.

.4. TcAChE concentrations

ATCh hydrolysis was measured at the final TcAChE concen-ration of 2 nM. Similar TcAChE concentrations were used for

easurements of BTCh inhibition of ATCh hydrolysis. The hydrol-sis of BTCh alone (at concentrations from 5 �M to 75 mM) wasollowed at 40 nM TcAChE, to provide good measurable rates. Inhe further analysis of the hydrolysis of BTCh by TcAChE, three dif-erent BTCh concentrations were used (50 �M, 0.5 mM, and 5 mM)t TcAChE concentrations from 5 nM to 1 �M.

.5. ATCh hydrolysis after various times of BTCh hydrolysis bycAChE

To follow BTCh hydrolysis, some 20 times higher TcAChEoncentrations were needed. Under such conditions an unex-

ected time dependent activity decrease during the measurementas observed. To get additional information on this process weiluted, immediately, after 2.5 min, 7.5 min and 15 min, the reac-ion mixture 100 times, supplemented new amount of DTNB andetermined the initial rate at 0.5 mM final ATCh concentration.

actions 187 (2010) 128–134 129

2.6. Kinetic analysis

The raw experimental data for the time courses of productformation measured under various conditions were analyzed asfollows. The differential equations for the reaction mechanisms ini-tially suggested for DmAChE and later applied also to electric-eelAChE and TcAChE were implemented in a non-linear regressionprogram [10]. The kinetic constants and proportional factors wereevaluated by direct iterative fitting of a numerically solved sys-tem of differential equations to the experimental progress curves(see Appendix A for details). The analysis was carried out in threestages: (i) for ATCh hydrolysis by TcAChE, where the original seven-intermediate reaction scheme with seven kinetic constants wasused [11]; (ii) for BTCh inhibition of ATCh hydrolysis by TcAChE,where the enlarged reaction scheme allowed the interaction of bothof these ligands with the peripheral anionic site (PAS) and the cat-alytic anionic site (CAS) of TcAChE, although only ATCh was allowedto yield products (Scheme 1 and [12]); (iii) for the hydrolysis ofBTCh by TcAChE, where the seven-intermediate reaction schemewas enlarged to allow a slow nonproductive blockade of TcAChE. Toachieve this, three additional intermediates were introduced (seeAppendix A).

3. Results

3.1. Hydrolysis of ATCh by TcAChE and its inhibition by BTCh

After examining the hydrolysis of ATCh and BTCh with identicalaliquots of the same TcAChE stock, it is clear that in comparisonwith the hydrolysis of ATCh, that of BTCh at 2 nM TcAChE over a timerange of a few minutes is negligible (Fig. 1). The hydrolysis of ATChby TcAChE can thus be examined in the absence and presence ofBTCh without the need to take into account the depletion of BTCh orits contribution to product formation. Fig. 2 shows the initial ratesfor the hydrolysis of ATCh by TcAChE in the absence and presenceof two fixed BTCh concentrations (0.5 mM and 5 mM). It needs to beemphasized here that these initial rates were determined througha simultaneous progress curve analysis of all of the available data.

Fig. 1. Comparison of the progress curves for the time courses of product formationfor the hydrolysis of ATCh and BTCh. TcAChE was used at 10 nM, and each of thesubstrates at 5 mM. Inset: expanded sweep time and hydrolysis measures. Note thevertical line following the Y axis for the hydrolysis of ATCh.

130 J. Stojan / Chemico-Biological Inter

Fa

ioKtfTo

3

Tttotabti1t

TCs

ig. 2. Characteristic pS curves for the hydrolysis of ATCh by TcAChE in the absencend presence of 0.5 mM and 5 mM BTCh.

nteractions of TcAChE with BTCh and the effects of this interactionn the hydrolysis of ATCh (see also Appendix A). The values of Kip,iL and KiLL represent the binding affinities of BTCh to the PAS andhe CAS of the free and the acetylated TcAChE. The proportionalactors ‘c’ and ‘d’, however, reflect the effects on the acetylation ofcAChE by ATCh and the deacetylation of TcAChE, both when BTChccupies the PAS.

.2. Hydrolysis of BTCh by TcAChE

To obtain good measurable activity for the hydrolysis of BTCh bycAChE, the TcAChE concentration was increased by 20-fold, andhe sweep time in each measurement was also increased by a fac-or of eight. Fig. 3A shows the progress curves of the time coursesf product formation for the hydrolysis of increasing concentra-ions of BTCh by 40 nM TcAChE. It can immediately be seen thatll of these progress curves are downwardly concave. This might

e expected for the progress curves at the low substrate concen-rations, where a considerable amount of the substrate, if not all,s hydrolyzed. However, at higher BTCh concentrations, less then0% of this substrate is consumed, and additionally, the bends inhe progress curves appear earlier in the time-course measure-

able 1haracteristic kinetic parameters for the hydrolysis of ATCh and BTCh by TcAChE, accortandard errors were fixed in the subsequent evaluations.

ATCh

Kp (�M) 461 ± 20 K(I)p (�M)KL 1.33 ± 0.08 K(I)L

KLL 160 ± 4 K(I)LL

k2 (s−1) 21,070 ± 723 k2 (s−1)k3 (s−1) 811 ± 2 k3 (s−1)a 1.0 ± 0.11 ab 0.025 ± 0.008 b

cI

dI

ki

k−i

KiL

Composite macroscopic kinetic constantsKiS = k−iKiL/

KS = KpKL 0.61 (mM) KS = KpKL

KSS = KpKLL 73.4 (mM) KSS = KpKLL

KLL/KL 120 KLL/KL

kcat 745 (s−1) kcat

Km 21.5 (�M) Km

kcat/Km 3.5 × 107 (s−1 M−1) kcat/Km

actions 187 (2010) 128–134

ments, and are then followed by linear product formation. Indeed,these bends occur at their earliest at intermediate BTCh concen-trations, while they occur later at the highest, and are not visibleat the lowest BTCh concentrations. To demonstrate these findingsunequivocally, we then followed BTCh hydrolysis at three fixedsubstrate concentrations, and varied the concentrations of TcAChEfrom 5 nM to as high as 1 �M (data not shown). At 50 �M BTCh, allof the progress curves followed simple, irreversible, second-orderconversion kinetics, suggesting that at low substrate concentra-tions the slow phase in the reaction is not seen. Subsequently, thevery same pattern as in Fig. 3A was seen at 0.5 mM and 5 mM BTCh,and the bend in the progress curves clearly occurred earlier at thehigher enzyme concentrations. Of note here, for the ATCh progresscurves that were analogous to those for BTCh shown in Fig. 3A (withexactly the same ATCh concentrations), these were straight lines,with concave curves only seen at low substrate concentrations, dueto the depletion of the ATCh substrate (data not shown).

3.3. ATCh hydrolysis after various times of BTCh hydrolysis byTcAChE

Sloped assymptotes of the progress curves for the hydrolysisof BTCh by TcAChE (Figs. 1 and 3A) display the establishment oflate steady-state in the reaction and, thus, clearly point to thereversible nature of the interaction. Consequently, the initial activ-ity should recover when diluting sufficiently the reaction mixture.Fortunately, 100 times dilution of the TcAChE–BTCh reaction mix-ture still allows for reliable activity determination using ATCh asa detection substrate. We, therefore, started the reaction betweenBTCh and TcAChE and tested the activity in this way, immediatelyand after 2.5 min, 7.5 min and 15 min. Surprisingly again, all curvesin these determinations were straight lines with similar slopes,suggesting instantaneous recovery after dilution (Fig. 4).

All of these findings from the experiments with ATCh alone,with ATCh inhibition experiments with BTCh, with BTCh aloneand recovery after BTCh reaction have been integrated into in areaction scheme (see Appendix A) that suggests the slow, non-productive establishment of a BTCh–TcAChE complex, where the

enzyme activity of TcAChE is completely blocked. The evaluationof the kinetic constants is in agreement with the interpretationthat the occurrence of this nonproductive complex is rare, but thatits dissociation rate is similar to that the productive BTCh–TcAChEcomplex, as suggested by recovery experiments.

ding to the reaction scheme given in Appendix A (Scheme 1). Parameters without

BTCh – substrate BTCh – inhibitor

28.7 28.7 ± 0.532.9 32.9 ± 2.118.2 18.2 ± 0.726.9 ± 0.1514 ± 33.40.2 ± 0.01

3.4 ± 0.10.016 ± 0.001

1283 ± 42583 ± 91.2 ± 0.08 × 10−4

ki 0.24 (mM)0.95 (mM)0.52 (mM)0.550.8 (s−1)27.8 (�M)2.8 × 104 (s−1 M−1)

J. Stojan / Chemico-Biological Interactions 187 (2010) 128–134 131

Fig. 3. (A) Progress curves for the time courses of product formation for the hydrolysis oconstants from Table 1. (B) Initial and steady-state rates for the hydrolysis of BTCh by TAppendix A and the constants from Table 1.

Fig. 4. Reactivation of TcAChE after 0 min, 2.5 min, 7.5 min and 15 min of preincu-bwf

4

rh[tapeasaA

times, and they again appear later at the concentrations of BTCh that

ation with BTCh. Measuring solution, containing 22 nM TcAChE and 5 mM BTCh,as diluted 100 times, ATCh (0.5 mM) and DTNB (0.66 mM) added and the product

ormation followed for 60 s.

. Discussion

Although AChEs are very specific enzymes, they can actuallyeact with many ligands. With the substrates in question, these areydrolyzed according to a general acid–base and covalent catalysis13,14]. To study the individual reaction steps in the catalytic cycle,here are several substrates to choose from: carboxylic alcohol ormide esters, carbamate quasi-substrates, organophosphorus com-ounds, and sulfonates. The first two of these substrate groups willventually be completely hydrolyzed, thus yielding the regener-

ted free enzyme. On the other hand, the organophosphates andulfonates are true catalytic poisons, the turnover of which stopst the level of the acylated enzyme. The fate such phosphorylatedChEs is dual: they can be ‘saved’ by special regenerating agents,

f BTCh by TcAChE. The theoretical curves were calculated using the correspondingcAChE. The data points were calculated using the corresponding equations from

called reactivators, or they will ultimately be lost in the process ofaging [1]. Sulfonylated AChEs, however, age as they are produced.

In the present study, the focus is on the reaction of TcAChE withBTCh. The structure of BTCh is very similar to that of the naturalsubstrate, ACh, although it is very poorly hydrolyzed by all ver-tebrate AChEs. The present study thus started with the commonprocedure, i.e. after establishing the negligible hydrolysis of BTChby TcAChE concentrations suitable for detecting ATCh hydrolysis(Fig. 1), BTCh was tested as an inhibitor of ATCh (Fig. 2). It turnsout that BTCh competes with ATCh at both of the substrate bindingsites of TcAChE, at the PAS and at the CAS. This was expected as X-ray data have revealed the exact positions of two BTCh moleculesin the gorge of the mouse AChE enzyme (PDB code 2HA4, [15]).

In the next step, the hydrolysis of BTCh itself was measured,by considerably increasing the concentration of TcAChE (20-fold),which achieved good measurable activities for product formation.Under these conditions, the time courses of product formation forthe hydrolysis of BTCh were of an unexpected shape, as shownin Fig. 3A. While the progress curves at low BTCh concentrationsapproached a plateau due to complete conversion of the substrate,the progress curves for higher BTCh concentrations showed down-wardly concave initial portions, and then later became sloped lines.In searching for an interpretation of this behavior, we extendedour measurements to three fixed BTCh substrate concentrations(50 �M, 0.5 mM, and 5 mM) with varying TcAChE enzyme concen-trations. Indeed, the progress curves at 50 �M BTCh all followedthe second-order, irreversible reaction mechanisms, ending stoi-chiometrically at the level where all of the added BTCh was used.Of note here, these progress curves would be different if a slowphase in the reaction had participated. It appears therefore thatat low substrate concentrations the reaction is completed beforethe slow phase would be expected. At intermediate substrate con-centrations, the bends in the progress curves appear at the earliest

cause substrate inhibition. Additionally, experiments with increas-ing enzyme concentrations show linear increases of the overallfirst-order ‘bending’ constant, i.e. the bends in the progress curvesappear earlier at the higher enzyme concentrations.

1 l Inter

ttwtA

wtstasgtbsnsatBi

cbchupsi

bciAwcwCicsstacsTaspBdswpr[otfisti

32 J. Stojan / Chemico-Biologica

One reason for slow decline of initial BTCh hydrolysis might behe fact that TcAChE is unusual among vertebrate AChEs in con-aining a free cysteine residue (Cys231). Its chemical modificationith sulfhydryl reagent DTNB, present in the assay, could influence

he hydrolysis profile, while it should not be affected in vertebrateChEs lacking such a cysteine near the active site gorge.

Although the late establishment of steady-state is consistentith the reversibility of the interaction between TcAChE and BTCh,

he recovery experiments could further uncover its nature. Namely,low binding reversible inhibitor or very poor substrate in its firsturnover with the enzyme in equimolar concentrations would givesimilar burst in the initial progress curve portion followed by a

loped assymptote. The reactivation kinetics in these cases wouldive concave upward progress curves, again with sloped assymp-otes, but usually with longer bends or the reaction would likelye irreversible if chemical modification of Cys231 were the rea-on. In our experiments, however, the enzyme and substrate wereever in equimolar concentrations and the progress curves mea-ured after dilution of TcAChE–BTCh reaction mixture, using ATChs the reporter substrate, were always linear and preincubationime independent (Fig. 4). Such a result indicates that the decline inTCh hydrolysis rate is rapidly reversed unlike the result expected

f a Cys231–TNB mixed disulfide were formed.With this information in hand, the absence of biphasic time

ourses in vertebrate AChEs without free cysteine would no longere a concern. Indeed, progress curves for the hydrolysis of BTCh byommercial electric-eel AChE (data not shown) and recombinantuman AChE (Rosenberry, personal communication), measurednder similar conditions, were linear. Thus, other differences inrimary and tertiary structure, rather than just the lack of corre-ponding cysteine should cause differences towards BTCh kineticsn vertebrate AChEs bearing between 60 and 70% identity.

After establishing these many characteristics essentially onlyy inspection and simple tests, they all had to be summarized in aomprehensive reaction scheme that would suggest the underly-ng molecular events. It is clear that such a scheme (as illustrated inppendix A) should include two parts: (i) an instantaneous section,hich immediately yields the products. This part is revealed by the

ompetition between the hydrolysis by TcAChE of ATCh and BTCh,ith BTCh as an inhibitor of ATCh hydrolysis at both the PAS andAS substrate binding sites and (ii) then there is a slowly occurring

nhibition of BTCh hydrolysis by BTCh itself. At the first glance, aonformational change would appear to be an obvious reason foruch slow homotropic inhibition, but nothing like this has beeneen in the resolved X-ray structure of mouse AChE [15]. Further,he curvature may result from a unique feature of TcAChE, namelyburied free sulfhydryl group that can be modified under some cir-umstances with a resulting change in enzyme activity. However,ulfhydryl modification, probably disulfide bond formation withNB, one half of DTNB, would be essentially irreversible but slopedsymptotes and instantaneous recovery with ATCh as a reporterubstrate do not support this hypothesis. Another plausible inter-retation would be a rarely occurring nonproductive binding of aTCh molecule to the PAS, followed by its instantaneous ‘gliding’own to the CAS in an orientation that is unfavorable for cataly-is. The subsequent binding of another BTCh molecule to the PASould then block the enzyme activity of the AChE. This latter inter-retation can be implemented in the existing seven-intermediateeaction scheme by the addition of only three new intermediates7,12, Appendix A]. Furthermore, a rarely occurring incorrect BTChrientation at the PAS should be represented by a low value of

he second-order association rate constant for this step. As therst-order dissociation rate constant should be similar to the corre-ponding constant when the substrate is accommodated correctly,he rare occurrence would be seen kinetically as a very low affin-ty. On the other hand, the affinity of TcAChE for BTCh at the CAS

actions 187 (2010) 128–134

should not differ much whatever orientation it is bound in, as longas that the main interaction occurs through the cation–PI interac-tions between the positive charge of BTCh and Trp84 of TcAChE[16].

It could be argued at this point that the slow step might be repre-sented by a low value of the first-order rate constants for the glidingof BTCh between the PAS and the CAS. This appears highly unlikely,because gliding down and up the active site gorge is largely one-dimensional diffusion, and the driving force of the positive chargeshould not be orientation dependent. Besides, the fictive concen-tration of improperly oriented substrate molecules (one in 105) isroughly equimolar with the enzyme concentration, thus intuitivelysuggesting that the binding, and not the gliding, is the slow step.

Strong confirmation for this hypothesis can be expected froma precise kinetic analysis. The instantaneous inhibition of ATChhydrolysis by BTCh can be evaluated using an appropriate initial-rate equation (see Appendix A and [7,12]), with the analysis ofcomplete progress curves considerably increasing the chances ofarriving at a global solution. Moreover, because the values of thekinetic constants for the hydrolysis of ATCh are known from pre-vious studies [11], only those that are characteristic of BTCh hadto be searched here. Therefore, the parameters of the numeri-cally solved system of differential equations for Scheme 1 (seeAppendix A) were fitted to the experimental data, using a non-linear regression program. Such an analysis is conducted underpurely nonequilibrium conditions, and it also takes into accountthe depletion of substrate and ligand during the course of thereaction. Consequently, an analogous progress curve analysis wasperformed with the data for the time courses of product formationduring the hydrolysis of BTCh by TcAChE, using the appropriatedifferential equations (see Appendix A). For the evaluation in thisstep, only five unknowns remained. These are the constants forthe acylation/deacylation events, and also two kinetic constantsfor describing the slow blockade: the second-order association rateconstant for the binding of BTCh to the PAS in a nonproductiveorientation, and its subsequent affinity constant for the CAS.

It should be noted once again that during the reaction of AChEswith their substrates, many concomitant events take place, addedto which, the occurrence of a slow phase further entangles the sit-uation. The interpretation here assumes that the slow homotropicinhibition of TcAChE by BTCh is a consequence of it producing ararely occurring unfavorable orientation at the PAS, which wouldthen lead to an apparently complete active-site blockade. All of thenecessary kinetic data have here been theoretically reproduced byintroducing only three additional steps in an operating reactionscheme, using a numerical solver.

Finally, it can thus be speculated that compounds with severalpossible nonproductive binding patterns will cause multi-phase,rather then only two-phase, time-resolved kinetics [17]. In contrast,if one compound adopts nonproductive binding in the active site ofan enzyme, this would by no means preclude the similar pattern inan active site residue mutant of the same species. This is particularlytrue for the enzyme from different source, because only long rangelinear progress curves for the hydrolysis of BTCh by electric-eel andhuman AChE could be recorded.

Conflict of interest statement

The author declares that there are no conflicts of interest.

Acknowledgements

The author wishes to thank Prof. Israel Silman for provid-ing a considerable amount of T. californica acetylcholinesterase,Prof. Terry Rosenberry for the measurements on human acetyl-

J. Stojan / Chemico-Biological Interactions 187 (2010) 128–134 133

S of two( acy) Ai consr

cat

A

ot

v

w

k

K

P

Q

h(ast

cheme 1. The kinetic reaction scheme that foresees all of the possible interactionsCAS: symbol on the right of E) sites of free (E and legacy) and acetylated (EA and legnhibitor (BTCh). Kp and Ki represent dissociation constants; k2 and k3 represent rateespectively.

holinesterase and Mrs. Nevenka Klenovsek-Spat for her technicalssistance. The work was supported by the Ministry of Science ofhe Republic of Slovenia, grant no. P1-0170.

ppendix A.

Analysis of the data for the hydrolysis of ATCh in the presencef BTCh. The scheme allows for the binding of both ligands to bothhe PAS and CAS substrate binding sites.

The derived initial rate equation is as follows:

= kcat[E]0[S][S] + Km

here

cat = k2k3

k2P + k3Q

m =

k3KpKL(1 + ((I)/KI) + ((I)/KIKiL)+ ((I)2/K2

I KiL))/(1 + b([S]/Kp) + d([I]/KI))k2P + k3Q

=

1 + ([S]/Kp) + ([S]/KpKLL) + ([S]2/K2p KLL) + ([S][I]/KpKLLKI)

+ ([I]/KI) + ([I]/KIKiLL) + ([I]2/KIKiLL) + ([S][I]/KpKIKiLL)

1 + a([S]/Kp) + c([I]/KI)

= 1 + KL + ([S]/Kp) + ([I]/KI) + KL([I]/KIKiL)1 + b([S]/Kp) + d([I]/KI)

For the analysis of the initial and steady-state rates for the

ydrolysis of BTCh by TcAChE, Scheme 1 is reduced to include sevenSE, ES, SES, EA, SEA, EAS, and SEAS) or ten (the seven plus IE, EI,nd SEI) intermediates, respectively. In such a reduced scheme ‘I’hould be substituted by ‘S*’. Indeed, the concentration is alwayshe concentration of the added substrate, i.e. BTCh.

ligands with the peripheral (PAS: symbol on the left of E with index p) and catalyticChE, with the conversion of only one ligand. E, AChE; S, substrate (ATCh or BTCh); I,

tants; and KL , KLL , KiL , KiLL and a–d are dimensionless ratios and proportional factors,

The corresponding equations for the initial rates are:

Km = (k3KpKL)/(1 + b([S]/Kp))k2P + k3Q

P = 1 + ([S]/Kp) + ([S]/KpKLL) + ([S]2/K2p KLL)

1 + a([S]/Kp),

Q = 1 + KL + ([S]/Kp)1 + b([S]/Kp)

And for the steady-state rates they are:

Km = k3KpKL(1 + ((I)/KI) + ((I)/KIKiL))/(1 + b([S]/Kp))K2P + k3Q

P =1 + ([S]/Kp) + ([S]/KpKLL) + ([S]2/K2

p KLL)+ ([S][I]/KpKLLKI) + ([I]/KI)

1 + a([S]/Kp),

Q = 1 + KL + ([S]/Kp) + ([I]/KI) + KL([I]/KIKiL)1 + b([S]/Kp)

References

[1] T.L. Rosenberry, Acetylcholinesterase, Adv. Enzymol. Relat. Areas Mol. Biol. 43(1975) 103–218.

[2] M. Harel, J.L. Sussman, E. Krejci, S. Bon, P. Chanal, J. Massoulié, I. Silman,Conversion of acetylcholinesterase to butyrylcholinesterase: modeling andmutagenesis, Proc. Natl. Acad. Sci. U.S.A. 89 (22) (1992) 10827–10831.

[3] D.E. Moore, G.P. Hess, Acetylcholinesterase-catalyzed hydrolysis of an amide,Biochemistry 14 (11) (1975) 2386–2389.

[4] I.W. Chubb, A.J. Hodgson, G.H. White, Acetylcholinesterase hydrolyzes sub-

stance P, Neuroscience 5 (12) (1980) 2065–2072.

[5] J.L. Johnson, B. Cusack, M.P. Davies, A. Fauq, T.L. Rosenberry, Unmasking tan-dem site interaction in human acetylcholinesterase. Substrate activation witha cationic acetanilide substrate, Biochemistry 42 (18) (2003) 5438–5452.

[6] J. Stojan, Kinetic evaluation of multiple initial rate data by simultaneous anal-ysis with two equations, Chem. Biol. Interact. 175 (1–3) (2008) 242–248.

1 l Inter

[

[

[

[

[

[

[16] J.L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman,Atomic structure of acetylcholinesterase from Torpedo californica: a prototypicacetylcholine-binding protein, Science 253 (1991) 872–879.

34 J. Stojan / Chemico-Biologica

[7] J. Stojan, L. Brochier, C. Alies, J.P. Colletier, D. Fournier, Inhibition of Drosophilamelanogaster acetylcholinesterase by high concentrations of substrate, Eur. J.Biochem. 271 (7) (2004) 1364–1371.

[8] J. Stojan, M. Zorko, Kinetic characterization of all steps of the interactionbetween acetylcholinesterase and eserine, Biochim. Biophys. Acta 1337 (1)(1997) 75–84.

[9] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, A new and rapid col-orimetric determination of acetylcholinesterase activity, Biochem. Pharmacol.7 (1961) 88–95.

10] J. Stojan, Analysis of progress curves in an acetylcholinesterase reaction:a numerical integration treatment, J. Chem. Inf. Comput. Sci. 37 (1997)1025–1027.

11] J.P. Colletier, D. Fournier, H.M. Greenblatt, J. Stojan, J.L. Sussman, J. Zaccai, I.Silman, M. Weik, Structural insights into substrate traffic and inhibition inacetylcholinesterase, EMBO J. 25 (12) (2006) 2746–2756.

12] I.B. Wilson, Mechanism of enzymic hydrolysis. I. Role of the acidic group inthe esteratic site of acetylcholinesterase, Biochim. Biophys. Acta 7 (3) (1951)466–470.

[

actions 187 (2010) 128–134

13] I.B. Wilson, Mechanism of hydrolysis. II. New evidence for an acylated enzymeas intermediate, Biochim. Biophys. Acta 7 (4) (1951) 520–525.

14] Y. Bourne, Z. Radic, G. Sulzenbacher, E. Kim, P. Taylor, P. Marchot, Substrateand product trafficking through the active center gorge of acetylcholinesteraseanalyzed by crystallography and equilibrium binding, J. Biol. Chem. 281 (39)(2006) 29256–29267.

15] O. Fekonja, M. Zorec-Karlovsek, M. El Kharbili, D. Fournier, J. Stojan, Inhibitionand protection of cholinesterases by methanol and ethanol, J. Enzyme Inhib.Med. Chem. 22 (4) (2007) 407–415.

17] P. Masson, M.T. Froment, E. Gillon, F. Nachon, S. Darvesh, L.M. Schopfer, Kineticanalysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides, Biochim.Biophys. Acta 1774 (9) (2007) 1139–1147.