Báo cáo khoa học: Kinetics of inhibition of acetylcholinesterase in the presence of acetonitrile doc

Prototypes of AChE inhibitors known to bind at the active site, PAS or both sites simultaneously are tacrine, gallamine and donepezil Fig.. The interaction of the inhibitors with AChE fr

presence of acetonitrile

Markus Pietsch, Leonie Christian, Therese Inhester, Susanne Petzold and Michael Gu¨tschow

Pharmaceutical Chemistry I, Pharmaceutical Institute, University of Bonn, Germany

Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine

hydrolase [1], which belongs to the a⁄ b hydrolase

family [2,3] The enzyme hydrolyses a broad range of

ester and amide substrates, showing the highest

speci-ficity for acetylselenocholine, acetylthiocholine (ATCh)

and acetylcholine (ACh) [4] Substrate cleavage

proceeds via a two-step mechanism: acylation of the

enzyme, followed by deacylation involving a water

molecule [5–7] This process is mediated by the

cata-lytic triad Ser200–His440–Glu327 (Torpedo californica

AChE, TcAChE, numbering [8]) located within the

active site at the bottom of a 20 A˚ deep gorge Sub-strate binding is facilitated by another component of the active site, the anionic site, which is characterized

by several conserved aromatic residues, such as Trp84 and Phe330 These residues have been shown to inter-act with the quaternary ammonium groups of ACh or ATCh via cation–p interactions [7–12] Further stabilization of the quaternary moiety arises from an electrostatic interaction with the acidic side-chain of Glu199 [7,12] A second substrate-binding site, the peripheral anionic site (PAS), lies essentially on the

Keywords

acetylcholinesterase; enzyme kinetics;

gallamine triethiodide; hyperbolic mixed-type

inhibition; tacrine hydrochloride

Correspondence

M Pietsch, School of Chemistry & Physics,

The University of Adelaide, Adelaide, SA

5005, Australia

Fax: +61 8 8303 4358

Tel: +61 8 8303 5360

E-mail: markus.pietsch@adelaide.edu.au

(Received 15 August 2008, revised 10

January 2009, accepted 11 February 2009)

doi:10.1111/j.1742-4658.2009.06957.x

The hydrolysis of acetylthiocholine by acetylcholinesterase from Electro-phorus electricus was investigated in the presence of the inhibitors tacrine, gallamine and compound 1 The interaction of the enzyme with the sub-strate and the inhibitors was characterized by the parameters KI, a¢, b or b,

Km and Vmax, which were determined directly and simultaneously from nonlinear Michaelis–Menten plots Tacrine was shown to act as a mixed-type inhibitor with a strong noncompetitive component (a¢  1) and to completely block deacylation of the acyl-enzyme In contrast, acetylcholin-esterase inhibition by gallamine followed the ‘steric blockade hypothesis’, i.e only substrate association to as well as substrate⁄ product dissociation from the active site were reduced in the presence of the inhibitor The rela-tive efficiency of the acetylcholinesterase–gallamine complex for the cataly-sis of substrate conversion was determined to be 1.7–25% of that of the free enzyme Substrate hydrolysis and the inhibition of acetylcholinesterase were also investigated in the presence of 6% acetonitrile, and a competitive pseudo-inhibition was observed for acetonitrile (KI= 0.25 m) The interac-tion of acetylcholinesterase with acetonitrile and tacrine or gallamine resulted in a seven- to 10-fold increase in the KIvalues, whereas the princi-pal mode of inhibition was not affected by the organic solvent The deter-mination of the inhibitory parameters of compound 1 in the presence of acetonitrile revealed that the substance acts as a hyperbolic mixed-type inhibitor of acetylcholinesterase The complex formed by the enzyme and the inhibitor still catalysed product formation with 8.7–9.6% relative efficiency

Abbreviations

ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; AP2238, 3-(4-{[benzyl(methyl)amino]methyl}phenyl)-6,7-dimethoxy-2H-2-chromenone; ATCh, acetylthiocholine; Ab, amyloid-b; MeCN, acetonitrile; Nbs 2 , 5,5¢-dithiobis(2-nitrobenzoic acid); PAS, peripheral anionic site; Tc, Torpedo californica.

surface of AChE [8] and consists of five residues,

Tyr70, Asp72, Tyr121, Trp279 and Tyr334, clustered

around the entrance of the active site gorge [13–17]

PAS binds ACh transiently as the first step in the

cata-lytic pathway, enhancing the catacata-lytic efficiency by

trapping the substrate on its way to the active site, and

allosterically modulates catalysis [7,12,18–22]

The principal physiological function of AChE,

mediated by the active site of the enzyme, is the rapid

hydrolysis of the neurotransmitter ACh at cholinergic

synapses and neuromuscular junctions, resulting in

the termination of the nerve impulse In addition to

this ‘classical’ function, several ‘nonclassical’ activities

of AChE have been reported, which are associated

with PAS [9,21,23,24] AChE is involved in neurite

growth [25], haematopoiesis and osteogenesis [26],

and acts as an adhesion protein in synaptic

develop-ment and maintenance [9] AChE has also been

shown to promote the pathophysiological assembly of

the amyloid-b (Ab) peptide into amyloid fibrils

in vitro [27,28] and in vivo [29,30], with complexes of

AChE and Ab displaying an enhanced neurotoxicity

in comparison with fibrils formed by Ab alone

[31–33] AChE has been found to be associated with

amyloid plaques and neurofibrillary tangles, two

hallmarks of Alzheimer’s disease (AD), and may

con-tribute to their development [34–36] A third

charac-teristic symptom of AD is the decrease in cholinergic

neurons, which causes a loss of cholinergic

neuro-transmission and may be responsible for the common

signs of memory failure [37,38] This ‘cholinergic

hypothesis’ provided the rationale for the current

major therapeutic approach to AD: the inhibition of

the catalytic function of AChE, thereby increasing the

bioavailability of ACh at the synaptic cleft, resulting

in an improvement in cholinergic neurotransmission and cognitive function [38–40]

With regard to the involvement of PAS in the pro-cesses of AD, the use of PAS inhibitors and dual-site inhibitors of AChE allows for the inhibition of the cat-alytic activity of the enzyme and also lowers the inci-dence of Ab fibril assembly [33,41,42] Prototypes of AChE inhibitors known to bind at the active site, PAS

or both sites simultaneously are tacrine, gallamine and donepezil (Fig 1), respectively The crystal structures

of each complexed with AChE have been published [10,41,43] In a previous study, we described 7-benzyl- 5,6,7,8-tetrahydro-2-isopropylamino-4H-pyrido[4¢,3¢:4,5]-thieno[2,3-d][1,3]thiazin-4-one (compound 1) (Fig 1), which inhibits AChE in the submicromolar range Kinetic analysis and structural similarities between donepezil, AP2238 (Fig 1) and compound 1 suggest that these substances act as dual-site inhibitors of AChE and bind along the active site gorge [42–44]

On the basis of these results, we performed a detailed kinetic study with the prototype inhibitors tacrine and gallamine, as well as compound 1 The interaction of the inhibitors with AChE from Electrophorus electricus was characterized using the kinetic models of AChE inhibition, shown in Scheme 1 [45,46] and Scheme 3 [47], as well as the simplified model for hyperbolic mixed-type inhibitors (Scheme 2), i.e general modifiers [48–50] The analysis presented herein allowed for the simultaneous determination of the kinetic parameters

KI, a¢, b or b, Km and Vmax directly from nonlinear Michaelis–Menten plots Recently, the interaction of gallamine and tacrine with AChE was found to be dependent on the presence of acetonitrile (MeCN) [51],

a cosolvent frequently used in AChE inhibition assays [44,51–54] In our ongoing investigations, this finding

Tacrine hydrochloride

N

NH2

x HCl

Gallamine triethiodide

O

O O N

N

N

3 I

S

S O

H N

1

N

Donepezil

O O

O

N

AP2238

O O

Fig 1 Inhibitors of AChE.

was analysed in detail by determining the effect of

MeCN on the kinetic parameters of AChE inhibition

Results and Discussion

Characterization of AChE inhibition and

estimation of the inhibitory parameters

The inhibition of AChE from E electricus by tacrine,

gallamine and compound 1 was determined

spectro-photometrically in a coupled assay with the substrate

ATCh and 5,5¢-dithiobis(2-nitrobenzoic acid) (Nbs2)

Inhibition studies were performed in the absence and

presence of 6% v⁄ v MeCN at various concentrations of

both the substrate [S] and the inhibitor [I] For

compar-ison, IC50 values were initially determined at a

sub-strate concentration of 500 lm by plotting the rates

versus [I] The inhibitory constants obtained for tacrine

(IC50= 0.047 ± 0.001 lm, no MeCN; IC50= 0.34 ± 0.02 lm, 6% MeCN) and gallamine (IC50= 1100 ±

60 lm, no MeCN; IC50= 2930 ± 140 lm, 6% MeCN) were in good agreement with results from a previous study [51] In the case of compound 1, AChE inhibition was only determined in the presence of 6% MeCN because of a lack of solubility in the absence of

an organic cosolvent As the enzyme was not completely inhibited at high concentrations of compound 1, residual activity at infinite concentration

of the inhibitor (v[I]fi ¥) had to be considered Using Eqn (15) (see Experimental procedures), values of

IC50= 0.58 ± 0.02 lm and v[I]fi ¥= 0.094 ± 0.004 (relative to the activity without inhibitor v0) were deter-mined, which confirmed previously reported data [44]

To characterize the inhibition of AChE, a kinetic model was considered (Scheme 1), which is analogous

to that introduced by Barnett and Rosenberry [45] and Szegletes et al [46] In this model, the substrate S binds to the enzyme E to form an initial enzyme–sub-strate complex, also called Michaelis complex ES [55] This complex proceeds to an acylated enzyme interme-diate EA, with the acylation rate constant k2, under simultaneous formation of the first product P1 The acyl-enzyme is then hydrolysed with the deacylation rate constant k3to give the second product P2and the free enzyme, which enters a new catalytic cycle If ATCh is used as substrate, thiocholine and acetate are formed as P1 and P2, respectively The Michaelis con-stant Kmand the maximal velocity Vmax, which can be experimentally determined, are expressed by Eqns (1) and (2), respectively [56,57]:

Km ¼ kSþ k2

kS 1þ k2

k3

1þ k2

k3

Vmax ¼ k2½E0

1þ k2

k3

where [E]0 is the total enzyme concentration and KSis the dissociation constant of ES The parameter kcat is equal to the quotient Vmax⁄ [E]0[46] For the hydrolysis

of ATCh by AChE from E electricus, values of the rate constants k2 (1.23· 106min)1) and k3 (9.3· 105min)1) have been obtained previously by direct measurements of the acetyl-enzyme As k2 is only about 1.3 times larger than k3, both constants are rate influencing [58]

An inhibitor I can bind to each of the three enzyme species to form a binary enzyme–inhibitor complex EI,

or the ternary complexes ESI and EAI with the

E

+

+

I

S

ESI

EI + P2 EI

kSI

+

S

+ I

k–I

kI

k–S

k–SI

EA

EAI

+ I

k–AI

kAI

k–S2

H2O

H 2 O

+

P1

P1

+

Scheme 1 Kinetic model of AChE inhibition.

E

+

+

I

EI + P2

E + P2

EI

kS

bk3

P1

k–I

k–S

EA

EAI

+ I

k–AI

H2O

H 2 O +

Scheme 3 Kinetic model of AChE inhibition excluding the

for-mation of ESI.

E

+

+

I

S

ESI'

EI + P

EI

+

S

α 'Ks

α 'KI

β 'kP

+ I

Scheme 2 Simplified kinetic model of the general modifier

mechanism.

enzyme–substrate complex and the acyl-enzyme,

respectively [46] The EI complex is capable of

bind-ing S, and catalyses product formation via ESI and

EAI, with the parameters a and b describing the

fac-tors by which k2 and k3 are altered As a general

steady-state solution for the reaction rate v in

Scheme 1 (based on extension of the

Michaelis–Men-ten expression) is too complex for useful comparison

with experimental data [45,46,48,59], a virtual

equilib-rium has been assumed for all reversible reactions in

Scheme 1 (i.e k)S k2, k)S2 ak2, k)SI k2,

k)SI ak2, k)AI k3, k)AI bk3) [45,46,59] The

resulting expression for v is given by Eqn (3) with the

dissociation constants KX expressed by the quotients

k)X⁄ kX

A direct analysis of AChE inhibition using Eqn (3)

is not possible, as contributions from the inhibition of

both acylation and deacylation complicate the

interpre-tation of the data In addition, estimates for a and b

are not separately available Therefore, the parameters

a and b were introduced to facilitate calculation At

saturating concentrations of I, these parameters are

expressed by Eqns (4) and (5), respectively [46]:

a ¼ aKI

KSI

¼ aKS

KS2

ð4Þ

b ¼ ab kð 2þ k3Þ

ak2þ bk3

ð5Þ

Under these conditions, i.e [I] fi ¥, S exclusively

binds to the EI complex and is converted to the

pro-ducts via ESI and EAI The reaction rate, v[I]fi ¥, at a

given substrate concentration is defined by a

combina-tion of Eqns (3–5):

v½I!1 ¼ bVmax½S

b

aKm þ ½S

ð6Þ

The kinetic parameters KI, a and b are usually

deter-mined by linearization of the Michaelis–Menten

equa-tion (Eqn 3) on the basis of the Lineweaver–Burk plot

and replotting of the slopes and intercepts obtained

(after normalization) [46,59] However, it was shown

that an iterative nonlinear optimization based on the

hyperbolic expression for v is, in general, more advan-tageous, as this method includes no transformation of the primary data, lower standard deviations and less bias in parameter estimates compared with algorithms using linearized plots [60] To apply such a nonlinear optimization, we simplified the kinetic model outlined

in Scheme 1 to that of the general modifier mechanism (Scheme 2) [48] In this model, ES and EA are not considered separately, but summed in a complex ES¢ that includes all enzyme–substrate intermediates In an analogous manner, ESI¢ represents the complexes of I with the substrate-bound enzyme and the acyl-enzyme [14] The dissociation constants of S from these binary and ternary complexes are KS= k)S⁄ kS and a¢KS= k)S2⁄ kS2, respectively [49] Both ES¢ and ESI¢

are capable of product formation (with P being both thiocholine and acetate [14]), governed by the catalytic constants kP and b¢kP, respectively The parameter b¢ reflects the efficiency of hydrolysis of ESI¢ compared with that of ES¢ This type of inhibition (b¢ > 0) is referred to as hyperbolic inhibition, as the shape of a reciprocal velocity 1⁄ v versus [I] plot is hyperbolic In contrast, a value of b¢ = 0 causes a linear dependence

of 1⁄ v on [I], and thus the inhibition is called linear [49,50] The dissociation constant of EI is KI= k)I⁄ kI [49] and thus defined as in Scheme 1, whereas a¢KI reflects a composite of constants for inhibitor binding

to the enzyme–substrate complex and the acyl-enzyme [14] The factor a¢ corresponds to the ratio of a¢KIand

KI As the overall equilibrium constant for the forma-tion of ESI¢ must be the same regardless of a path via ES¢ or EI, the same factor a¢ must be included in the model [49,61] Derivation of the general velocity equa-tion for the system in Scheme 2 can be performed assuming a rapid equilibrium or steady-state condition The first method gives a relatively simple expression, whereas the steady-state approach results in a very complex expression containing squared [S] and [I] terms However, the steady-state velocity equation simplifies to the same form as the rapid equilibrium velocity equation when pseudo-equilibrium conditions prevail (i.e k)S kP), as, in this case, the Michaelis constant Km= (k)S+ kP)⁄ kS substitutes for KS=

k)S⁄ kSin the velocity equation [49,61,62] In addition,

Km

1þ



I

KI

1þa



I

KSI

0

B

@

1 C

A þ ½S k2kþ k3 3



I

KSI

1þa



I

KSI

0 B

@

1 C

A þ k2kþ k2 3



I

KAI

1þb



I

KAI

0 B

@

1 C A

0 B

@

1 C A

ð3Þ

Kmhas been found to be similar to KSfor several

sub-strates of AChE [63] For the purposes of the present

study, a rapid equilibrium and an irreversible character

of the catalytic step were assumed Under these

condi-tions, the Michaelis–Menten equation for the general

type of inhibition shown in Scheme 2 is as follows:

Km

1þ



I

KI

1þb

0

I

a0KI

0 B

@

1 C

A þ ½S

1þ



I

a0KI

1þb

0

I

a0KI

0 B

@

1 C A ð7Þ

At saturating concentrations of I, the products are

exclusively formed from ESI¢ with a rate constant b¢kP

Under these conditions, the rate v[I]fi ¥ can be

expressed by Eqn (8) [44,49]:

v½I!1 ¼ b

0

Vmax½S

As the rate v[I] fi ¥ must be the same, regardless of

whether the model in Scheme 1 or 2 is applied, Eqns (6)

and (8) can be set as equal Therefore, the parameters a

and b in Eqn (6) can be expressed by means of a¢ and b¢

as follows: b = b¢ and a = b¢ ⁄ a¢ = b ⁄ a¢ In addition,

the value KI is equally defined in both kinetic models

(Schemes 1 and 2); thus, all three parameters

character-izing the inhibition according to the kinetic model in

Scheme 1, i.e KI, a and b, can be determined on the

basis of the simplified model depicted in Scheme 2 This

methodology was applied to the inhibition of AChE by

gallamine (in the absence of MeCN) and compound 1

(with 6% MeCN) The PAS inhibitor gallamine

(with-out MeCN) has already been reported to follow the

kinetic model in Scheme 1 [46], whereas inhibition by

compound 1 was found not to be complete at saturating

concentrations of I, i.e v[I] fi ¥ and therefore b are

greater than zero

For inhibitors attacking the active site of AChE and

containing a positively charged quaternary nitrogen

atom, it has been reported that they act not only by

binding to the free enzyme at the same site as the

sub-strate, but also by adding to the acyl-enzyme However,

these compounds do not inhibit through attachment to

the Michaelis complex [47,55,64–66] An example of

such an inhibitor is tacrine, which has been shown to

occupy the anionic binding site of TcAChE by being

sandwiched between the aromatic rings of Trp84 and

Phe330, mainly through p–p interactions and cation–p

interactions In addition, a direct hydrogen bond is

formed between the acridinic protonated nitrogen of the

inhibitor and the carbonyl oxygen of His440 [10]

Crys-tallographic studies on AChE complexed with ACh and

ATCh, as well as the nonhydrolysable substrate ana-logue 4-oxo-N,N,N-trimethylpentanaminium [7,12], revealed that these compounds also interact with Trp84 and Phe330 (TcAChE numbering [8]) Thus, it is unli-kely that tacrine binds to the Michaelis complex, i.e no ternary complex ESI is formed However, in the crystal structure of AChE with tacrine, the immediate vicinity

of the catalytic serine is not occupied by the inhibitor [10], and thus tacrine is probably able to bind to the EA complex [67] Under these conditions, the kinetic model

in Scheme 1 can be simplified to that shown in Scheme 3 As I does not interact with ES to form ESI, the value of the dissociation constant KSI in Scheme 1 becomes very large and the quotient KI⁄ KSIis virtually zero Equation (4) reveals that the ratios KI⁄ KSI and

KS⁄ KS2are equal, and thus the dissociation constant KS2 must also become very large (i.e the formation of ESI does not occur via binding of S to EI) An identical model as depicted in Scheme 3 has been used by Krupka and Laidler [47] to explain AChE inhibition caused by the interaction of I with E and EA The Michaelis–Men-ten equation for this type of inhibition is obtained by simplification of Eqn (3) with KSI fi ¥:

Km 1þ



I

KI

þ ½S k3

k2þk3

þ k2

k2þk3



I

KAI

1þb

I

KAI

0 B

@

1 C A

0 B

@

1 C A ð9Þ

At saturating concentrations of I, the rate v[I] fi ¥ is equal to zero when calculated using Eqn (9) This means that the kinetic model in Scheme 3 and Eqn (9) are only applicable if complete inhibition occurs

In analogy with the kinetic model in Scheme 2, the dissociation constant KAI, obtained from Scheme 3, was termed a¢KI with a¢ corresponding to the ratio of a¢KI and KI Additional rearrangement of Eqn (9) results in the following expression for v:

Km 1þ



I

KI

þ ½S

1þ



I

1þ bk3

k2

a0KI 1þ k3

k2

1þ b

I

a0KI

0 B B B B

1 C C C C ð10Þ

Equation (10) was used to analyse AChE inhibition by tacrine in the absence and presence of 6% MeCN

In the present study, we applied an iterative nonlinear optimization based on the hyperbolic Michaelis–Menten

Eqn (7) (with b¢ = b) and Eqn (10) to calculate the

parameters KI, a¢, b (Eqn 7) or b (Eqn 10), Kmand Vmax

simultaneously from plots of rate versus [S] in the

presence of various inhibitor concentrations However,

provisional estimates of the kinetic parameters were

necessary prior to the computer-assisted iterative

deter-mination [60] To obtain such estimates for Km and

Vmax, we determined these parameters separately for

each set of data (tacrine without MeCN, tacrine with

MeCN, gallamine without MeCN, gallamine with

MeCN, and compound 1 with MeCN) in the absence of

inhibitor The data were analysed by a nonlinear

regres-sion according to Eqn (11), which represents a

simplifi-cation of Eqn (3) with [I] = 0:

v ¼ Vmax½S

Using this method, the following values of Km and

Vmax were calculated for the five sets of data: tacrine,

no MeCN: Km= 101 ± 14 lm, Vmax= 110 ± 3%;

tacrine, 6% MeCN: Km= 684 ± 41 lm, Vmax=

229 ± 5%; gallamine, no MeCN: Km= 135 ±

19 lm, Vmax= 116 ± 3%; gallamine, 6% MeCN:

Km= 671 ± 20 lm, Vmax = 235 ± 3%; compound

1, 6% MeCN: Km= 606 ± 32 lm, Vmax= 229 ±

4% (The rate of the AChE-catalysed hydrolysis of

500 lm ATCh, corrected by the value of the

nonenzy-matic hydrolysis, was set to 100% in all experiments.)

Provisional estimates for KI, a¢ and b in Eqn (7)

(b¢ = b) were obtained by analysing the data of the

AChE inhibition studies with the specific velocity plot

developed by Baici [61] This method is advantageous

over the commonly used Lineweaver–Burk plot or the

similar Hanes–Woolf plot [49], as it always gives linear

plots, independent of whether the inhibition is linear

or hyperbolic The type of inhibition can be obtained

by simple inspection of the specific velocity plot

(Eqn 12), and linear replots permit the calculation of

KI, a¢ and b [61] On the basis of Eqn (12), the

quo-tient of the rate without inhibitor and the rate in the

presence of inhibitor, v0⁄ vI, was plotted against

r⁄ (1 + r), with r being equal to [S] ⁄ Km (Doc S1,

Fig S1A,B, see Supporting information):

v0

vI

¼

1

a0KI

 1

KI

I

1þb



I

a0KI

r

1þ r þ

1þ



I

KI

1þb



I

a0KI

ð12Þ

To obtain provisional estimates for KI, a¢ and b in

Eqn (10), we developed a graphical method based on

Eqn (13), which is similar to the specific velocity plot (Doc S1, Fig S2A,B, see Supporting information):

v0

vI ¼

1þ



I

1þbk3 k2

a0KI 1þ k3 k2

1þb



I

a0KI



I KI

0 B B B B B

@

1 C C C C C A

r

1þ r þ 1þ



I KI

ð13Þ

Investigations of AChE inhibition by tacrine, in the absence and presence of 6% MeCN, on the basis of the modified specific velocity plot (Eqn 13, data not shown) indicated a mixed-type inhibition that tended

to noncompetitive inhibition in both cases with KI= 0.038 lm, a¢ = 0.91 and KI= 0.25 lm, a¢ = 1.0, respectively The value b was determined to be equal

to –0.004 for the enzyme–inhibitor interaction, both with and without MeCN As b < 0 cannot be defined

by the mechanism in Scheme 3, b was set to zero for the calculation of the kinetic parameters by Eqn (10) AChE inhibition by gallamine without MeCN and compound 1 in the presence of 6% MeCN, analysed according to Eqn (12) (Fig S1A, see Supporting infor-mation), was found to follow a hyperbolic mixed-type inhibition with a¢ > 1 and b > 0 This is shown for gallamine by the common intersection point of the lines

in the specific velocity plot at r⁄ (1 + r) > 1; v0⁄ vI= 1 (Fig S1A, see Supporting information), as well as by discrete intercepts of the replots (Fig S1B, see Support-ing information) [61] On the basis of these replots, the parameters KI= 330 lm, a¢ = 5.7 and b = 0.31 were determined Using this method, KI= 0.52 lm, a¢ = 1.3 and b = 0.077 were calculated for the inhibition of AChE by compound 1 (data not shown)

In contrast with the study without MeCN, a plot of

v0⁄ vIversus r⁄ (1 + r) for AChE inhibition by gallamine

in the presence of 6% MeCN showed an array of curves with a common intersection point close to r⁄ (1 + r) = 1; v0⁄ vI= 1 (Fig S2A, see Supporting information) A plot of int0⁄ (int0)1) versus 1 ⁄ [I], where int0 is the inter-cept on the ordinate axis [r⁄ (1 + r) = 0], and an initial analysis (Fig S2B, see Supporting information) gave an intercept equal to unity Such a behaviour indicates competitive inhibition [61], which is described by Eqn (14):

Km 1 þ



I

KI

þ ½S

ð14Þ

As an approximation, Eqn (14), a simplified form of

the Michaelis–Menten Eqns (3) and (10) valid for

com-petitive inhibitors, and an estimated value KI= 1130

lm, obtained on the basis of the modified specific

velocity plot (Eqn 13; Doc S1, Fig S2B, see

Support-ing information), were used to quantify the interaction

of AChE with gallamine in the presence of 6% MeCN

Determination of the parameters of inhibition

using the Michaelis–Menten equation

The final kinetic analysis of the inhibition by tacrine

(Fig 2A,B), gallamine (Fig 3A,B) and compound 1

(Fig 4) in the absence and presence of 6% MeCN was

accomplished using Eqn (7) (b¢ = b), Eqn (10) or

Eqn (14), as outlined above The rates of

enzyme-catalysed substrate cleavage were analysed as a

func-tion of both [S] and [I], and the parameters KI, a¢, b or

b, Km and Vmax were calculated simultaneously

(Table 1) Parameter estimates were taken from

(modi-fied) specific velocity plots and Michaelis–Menten plots

in the absence of inhibitor (see above) All the values

of Km and Vmax calculated independently by the

non-linear optimization of Eqns (7) and (10) (Table 1) were

in good agreement with the parameter estimates

obtained in the absence of the inhibitors (see above)

In the case of gallamine investigated in the presence of

MeCN, the calculation of the kinetic parameters was

based on Eqn (14), as a competitive mode of inhibition

was assumed from the modified specific velocity plot

(Fig S2A,B, see Supporting information) The Km

value of 778 lm obtained differed considerably from

the parameter estimate of 671 lm In contrast, the

cal-culated Vmax value of 245% was very similar to the

parameter estimate of 235% This behaviour can be

explained by the competitive mode of inhibition The

substrate affinity of the enzyme will be reduced, i.e

the apparent Km value will be increased, whereas the

maximum velocity of product formation Vmax is not

affected [49,50] With a given set of data for rates as a

function of [S], the determination of Vmaxand thus Km

becomes less accurate when [S] is low relative to the

apparent Kmvalue [49] This occurred in our study for

high concentrations of gallamine in the presence of 6%

MeCN

One possibility to avoid this accuracy problem

would be to investigate the enzyme–inhibitor

interac-tion in the presence of higher substrate concentrainterac-tions

However, in the case of AChE, it is known that

sub-strate inhibition arises under such conditions, with

PAS being involved in the mechanism [12,19,22,68–70]

As gallamine binds to PAS [41], a more complex mode

of inhibition might result [71] Therefore, we did not

use higher substrate concentrations to analyse AChE inhibition by gallamine in the presence of 6% MeCN, which might also have revealed a deviation from the apparent competitive inhibition Instead, we investi-gated the interaction of the inhibitor with AChE over

a range of [S], where substrate inhibition was not observed [7,14] The maximum [S] value of 2250 lm corresponded to  16–22Km for experiments without

0 500 1000 1500 2000 2500

0 25 50 75 100

125

A

B

0 500 1000 1500 2000 2500

0 40 80 120 160 200

Fig 2 Inhibition of AChE by tacrine in the absence (A) and pres-ence (B) of 6% MeCN Michaelis–Menten plots using mean values and standard deviations of rates from four separate experiments in

100 m M sodium phosphate, 100 m M NaCl, pH 7.3 with 350 l M Nbs 2 and 0.033 UÆmL)1AChE (A) Concentrations of tacrine were

as follows: open circles, [I] = 0; filled circles, [I] = 0.025 l M ; open squares, [I] = 0.05 l M ; filled squares, [I] = 0.1 l M ; open triangles, [I] = 0.15 l M ; filled triangles, [I] = 0.2 l M ; open reversed triangles, [I] = 0.25 l M Nonlinear regression according to Eqn (10) gave K I = 0.027 ± 0.003 l M , a¢ = 1.4 ± 0.2, K m = 101 ± 5 l M and Vmax=

110 ± 1% (B) Concentrations of tacrine were as follows: open circles, [I] = 0; filled circles, [I] = 0.125 l M ; open squares, [I] = 0.25 l M ; filled squares, [I] = 0.5 l M ; open triangles, [I] = 0.75 l M ; filled triangles, [I] = 1.0 l M ; open reversed triangles, [I] = 1.25 l M Nonlinear regression according to Eqn (10) gave K I = 0.26 ± 0.02 l M , a¢ = 1.1 ± 0.2, K m = 691 ± 35 l M and Vmax= 229 ± 4%.

In (A) and (B), the b values were set to zero, as the starting values obtained from the modified specific velocity plots (data not shown) were b < 0.

MeCN and 3.2–3.7Km for experiments with MeCN

(Table 1) To incorporate a more accurate Vmax value

in the fitting process, this parameter was set to a value

of 235%, obtained in the absence of gallamine (see

above) An analysis of the data according to Eqn (14)

using this set Vmaxvalue gave Km= 699 lm (Table 1)

This value is closer to the parameter estimate of

671 lm, as well as the Km values calculated for the

inhibition of AChE by tacrine and compound 1 in the presence of MeCN (Table 1) The KI value obtained using the predefined Vmax value in Eqn (14) was

2020 lm (Table 1), whereas an only slightly larger value of 2150 lm resulted when Vmax was determined independently

The determination of the factors a¢, b and b (Table 1) was performed to obtain an insight into the mode of inhibition, and the KI values (Table 1) pro-vided information on the inhibitory potency of the compounds As depicted in Schemes 2 and 3, the fac-tor a¢ defines whether the inhibifac-tor binds to an enzyme–substrate species (ES and EA combined together as ES¢ in Scheme 2 or EA in Scheme 3) with

a greater affinity than to the free enzyme, or vice versa The preference of the inhibitor for binding to an enzyme–substrate species is reflected in values where a¢ < 1, which indicate mixed-type inhibition with a pronounced uncompetitive component A higher affin-ity of the inhibitor to the free enzyme, seen where a¢ > 1, corresponds to mixed-type inhibition with a more competitive character A pure noncompetitive mode of inhibition is characterized by a¢ = 1, i.e an equal affinity of the inhibitor to any form of the enzyme

An investigation of AChE inhibition by tacrine in the absence of MeCN, according to the kinetic model

in Scheme 3, gave values of KI= 0.027 lm and

0 500 1000 1500 2000 2500

0

25

50

75

100

125

A

B

0 500 1000 1500 2000 2500

0

40

80

120

160

200

Fig 3 Inhibition of AChE by gallamine in the absence (A) and

pres-ence (B) of 6% MeCN Michaelis–Menten plots using mean values

and standard deviations of rates from four separate experiments in

100 m M sodium phosphate, 100 m M NaCl, pH 7.3 with 350 l M

Nbs2 and 0.033 UÆmL)1 AChE (A) Concentrations of gallamine

were as follows: open circles, [I] = 0; filled circles, [I] = 500 l M ;

open squares, [I] = 1000 l M ; filled squares, [I] = 2000 l M ; open

tri-angles, [I] = 3000 l M ; filled triangles, [I] = 4000 l M ; open reversed

triangles, [I] = 5000 l M Nonlinear regression according to Eqn (7)

gave K I = 270 ± 20 l M , a¢ = 15 ± 2, b¢ = b = 0.25 ± 0.03,

Km= 135 ± 8 l M and Vmax= 116 ± 1% A value of a = 0.017 was

calculated as the quotient of b and a¢ (B) Concentrations of

gall-amine were as follows: open circles, [I] = 0; filled circles,

[I] = 750 l M ; open squares, [I] = 1500 l M ; filled squares,

[I] = 3000 l M ; open triangles, [I] = 4500 l M ; filled triangles,

[I] = 6000 l M ; open reversed triangles, [I] = 7500 l M Nonlinear

regression according to Eqn (14) with V max being set to 235% gave

KI= 2020 ± 50 l M and Km= 699 ± 10 l M

0 500 1000 1500 2000 2500

0 40 80 120 160 200

Fig 4 Inhibition of AChE by compound 1 in the presence of 6% MeCN Michaelis–Menten plot using mean values and standard deviations of rates from four separate experiments in 100 m M sodium phosphate, 100 m M NaCl, pH 7.3 with 350 l M Nbs2 and 0.033 UÆmL)1AChE Concentrations of compound 1 were as fol-lows: open circles, [I] = 0; filled circles, [I] = 1.5 l M ; open squares, [I] = 3.0 l M ; filled squares, [I] = 4.5 l M ; open triangles, [I] = 6.0 l M ; filled triangles, [I] = 7.5 l M Nonlinear regression according to Eqn (7) gave K I = 0.59 ± 0.05 l M , a¢ = 1.1 ± 0.1, b¢ = b = 0.096 ± 0.007, K m = 607 ± 25 l M and V max = 230 ± 3%.

A value of a = 0.087 was calculated as the quotient of b and a¢.

a¢ = 1.4 (Table 1) The parameter b was necessarily

set to zero for the nonlinear analysis (Table 1) and a

catalytically inactive EAI (Scheme 3) could therefore

be concluded Thus, the deacylation of the acyl-enzyme

was completely blocked by the inhibitor A mixed-type

inhibition, tending more to noncompetitive inhibition,

was found with tacrine This was characterized by an

a¢ value close to unity, and indicated that the affinity

of tacrine towards AChE was only minimally affected

by acylation of the active site serine Such a kinetic

behaviour has been reported to occur only if b = 0

and the acylation rate constant of substrate conversion

k2 is equal to or larger than the deacylation rate

con-stant k3[47] Both requirements are fulfilled, as shown

by the present study and as reported by Froede and

Wilson [58], respectively

These findings were in agreement with the results of

Nochi et al [72] (obtained with ATCh and AChE from

E electricus), who reported KI= 20.4 nm and KI* =

a¢KI(1 + k3⁄ k2) = 38.3 nm for tacrine on the basis of

the kinetic model in Scheme 3 (with b = 0) [72,73] For

comparison, we calculated a¢ = 1.1 using these values of

KIand KI* This result also indicates a mixed-type

inhibi-tion with a pronounced noncompetitive component

Inhibition of AChE by gallamine (in the absence of

MeCN), analysed according to the kinetic model in

Scheme 2, was characterized by values of

KI= 270 lm, a¢ = 15 and b¢ = b = 0.25 A value of

a = 0.017 was calculated as the quotient of b and a¢

(Table 1) The a¢ value obtained demonstrated the

preference of gallamine to bind to the free enzyme

rather than to the enzyme–substrate species ES and

EA (combined in ESI¢, Scheme 2), which indicates a

mixed-type inhibition with a pronounced competitive

component Such a kinetic behaviour has recently been

reported by Mooser and Sigman [74], who found pure

competitive inhibition (KI= 140–320 lm) for the

interaction of gallamine with AChE from E electricus

The parameter b represents the substrate conversion catalysed by an inhibitor-bound enzyme species com-pared with that by the free enzyme (both Schemes 1 and 2) Our study found a value of b = 0.25 for the interaction of AChE with gallamine (Table 1), which confirmed the parameter estimate based on the specific velocity plot (see above) Both b and the calculated value of a were in agreement with the data obtained

by Szegletes et al [46] for the inhibition of AChE by gallamine with ATCh as substrate, who reported

b = 0.44 and a = 0.019 Under the assumption of equilibrium conditions, a is represented by Eqn (4) Therefore, low values of this parameter require either that ESI (Scheme 1) is not formed (KS⁄ KS2 and

KI⁄ KSI 0) or that a  0 (Scheme 1) As shown in our study, a depends on both b and a¢, with the rela-tively large value of the latter parameter indicating a comparably low affinity of both the substrate and the inhibitor to form an enzyme–substrate–inhibitor spe-cies (Scheme 2) Thus, we hypothesize that the low a value results from a diminished formation of ESI rather than from inhibition of acylation (Scheme 1) Recently, nonequilibrium analysis of AChE inhibition

by the PAS ligands propidium and gallamine resulted

in the construction of the ‘steric blockade hypothesis’ (based on the model in Scheme 1) This hypothesis demonstrates that PAS ligands inhibit substrate hydro-lysis without inducing conformational changes in the active site [46] Nonequilibrium conditions are charac-terized by k)S< k2 and k)S2< ak2 [4,46], and thus

a kS2⁄ kSwas concluded according to Szegletes et al [46] The ‘steric blockade hypothesis’ implies that a ligand bound to PAS slows down ligand entry into and exit from the active site of AChE (kS2< kS and

k)S2< k)S) without affecting the thermodynamics of the binding of active site-directed ligands (KS2= KS)

It also stipulates that the PAS ligand has no effect on the rate constants of substrate acylation and deacylation

Table 1 Inhibition of AChE from Electrophorus electricus Values with standard error were calculated using data (mean values) from four separate experiments, at five or six inhibitor concentrations and 10 or 12 substrate concentrations Analysis of the data was performed using Eqn (7) (gallamine triethiodide, no MeCN; compound 1, 6% v ⁄ v MeCN; b¢ = b), Eqn (10) (tacrine · HCl, no MeCN; tacrine · HCl, 6% v ⁄ v MeCN) or Eqn (14) (gallamine triethiodide, 6% v ⁄ v MeCN).

a A value of 1.3 for k2⁄ k 3 has been taken from the literature [58] to calculate a¢ for experiments with tacrine · HCl, no MeCN, and tacrine · HCl, 6% v ⁄ v MeCN b Starting value b < 0, thus b was set to zero c ND, nondeterminable d a = b ⁄ a¢ = 0.017 e b = 1 in Scheme 3, thus a¢ is nondeterminable f

V max was set to 235%, determined during provisional estimate investigations in the absence of gall-amine triethiodide g a = b ⁄ a¢ = 0.087.

(a = b = 1), and that bound substrate does not alter

the interaction of the PAS ligand with the enzyme

(kI= kSI= kAI and k)I= k)SI= k)AI) Thus, only

the ratio k)I⁄ kI, i.e the KI value, is relevant [46] As

an extension of the steric blockade model, it was

pro-posed that bound PAS ligands also reduce the

dis-sociation rate constants for product release from the

active site, which becomes rate limiting at high [S]

[19,46]

On the basis of the ‘steric blockade hypothesis’, we

concluded that the inhibition of AChE by gallamine

(in the absence of MeCN) decreased the rate constants

kS2 and k)S2 (Scheme 1) to values  1.7% of kS and

k)S in our experiments This conclusion agrees with

the result of a simulated gallamine inhibition of ATCh

hydrolysis under nonequilibrium conditions [46], where

kS2 and k)S2 were set to 1.5% of kS and k)S,

respec-tively, to obtain optimal correlation between the

calcu-lated and experimentally determined parameters KI, a

and b The last two parameters can also be used to

characterize the relative efficiency of EI to catalyse

substrate conversion: a is defined as the ratio of the

second-order rate constant kcat⁄ Km with saturating [I]

to that in the absence of inhibitor, and b is the

quo-tient of the first-order rate constants kcat for substrate

conversion by EI (at saturating [I]) and E (Scheme 1)

[46] According to Eqns (6) and (11), a and b represent

the relative efficiency of EI (Scheme 1) if [S] << Km

and [S] Km, respectively Thus, the efficiency of the

complex AChE–gallamine to hydrolyse ATCh is 1.7–

25% of that of free AChE

Influence of MeCN on the inhibition of AChE

The inhibition of AChE by tacrine, gallamine and

compound 1 was investigated in the presence of 6%

v⁄ v MeCN (corresponding to a concentration of

1.15 m), and the results are shown in Figs 2B,3B,4 and

Table 1 Even without the addition of inhibitors, our

research showed that the presence of MeCN reduced

the rate of enzyme-catalysed substrate conversion,

which is in accordance with several literature reports

on soluble and immobilized AChE from E electricus

[75–80] At the highest substrate concentration used in

our experiments, [S] = 2250 lm, the absolute enzyme

activity without MeCN was 0.251 ± 0.052 min)1

(n = 8), whereas the addition of 6% MeCN resulted

in a decrease in the rate to 0.089 ± 0.020 min)1

(n = 8), i.e to 36% (data not shown) As depicted in

Table 1, MeCN also had an influence on the Kmvalue

of enzymatic substrate conversion, which increased

from 101–135 to 607–699 lm when 6% MeCN was

present in the assay A similar result was found in a

study by Ronzani [76], where Km values of 85 and

750 lm were determined for the AChE-catalysed con-version of ATCh in the absence and presence of 6.5% MeCN, respectively The latter Km value was calcu-lated on the basis of a competitive mode of inhibition suggested for MeCN and KI= 0.16 m [75,76] For competitive inhibitors, KI can be calculated according

to the equation KI= [I]⁄ [(Km¢ ⁄ Km))1], where Km¢ is the Michaelis constant in the presence of a certain amount of inhibitor [75,76] Applying this equation to our experiments and using mean values of the data shown in Table 1 (Km¢ = 666 lm at [MeCN] = 1.15 m, Km= 118 lm without MeCN), we calculated

an equivalent KIvalue of 0.25 m for MeCN Consider-ing the cosolvent MeCN as a competitive inhibitor, it might be included as a ‘second’ inhibitor in the fitting equations to analyse the influence of the ‘first’ inhibi-tor (i.e tacrine, gallamine or compound 1) Such attempts have, however, not been made in this study The inhibition experiments performed in the pres-ence of 6% MeCN and tacrine or 6% MeCN and gall-amine were analysed according to the kinetic model in Scheme 3, as ESI was assumed not to be formed in both cases (see above) Our investigations on the basis

of Eqns (10) and (14) revealed increased KI values for the two inhibitors compared with the studies without MeCN For tacrine and gallamine, 9.6-fold and 7.5-fold increases in the dissociation constant were observed, which resulted in KI= 0.26 lm and

KI= 2020 lm, respectively (Table 1) The factors for the increase in KI are in good agreement with data from a previous study [76], where a sevenfold increase

in KI was determined for the inhibition of AChE by neostigmine iodide in the presence of 6.5% MeCN and [S] = 20Km In addition, a considerable loss of inhibi-tion of immobilized AChE by dichlorvos and parao-xon in the presence of increasing amounts of MeCN (0–15%) was reported [79] This loss was suggested to

be caused by the denaturing effect of the organic solvent [79,81], which can also be considered as a pseudo-inhibition process [80] One reason for enzyme denaturation in the presence of water-miscible solvents, such as MeCN, might be the removal of essential water molecules from the enzyme, necessary for mani-festing the catalytic activity [80,82]

The inhibition of AChE by tacrine was characterized

by values of a¢ = 1.1 and b = 0 (Table 1), i.e tacrine (in the presence of 6% MeCN) acts as a mixed-type inhibitor with a strong noncompetitive component and completely blocks deacylation of EAI (Scheme 3) This behaviour is identical to that in the absence of MeCN

In contrast with tacrine, the PAS ligand gallamine tends to inhibit AChE in a competitive manner During