Tài liệu Báo cáo khoa học: High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine pptx

In human BuChE, the corresponding residue is N2.] This aspartate, part of the peripheral anionic site, contributes to the affinity of positively charged substrates for the active site, an

High activity of human butyrylcholinesterase at low pH

in the presence of excess butyrylthiocholine

Patrick Masson1, Florian Nachon1,2, Cynthia F Bartels2, Marie-Therese Froment1, Fabien Ribes1,

Cedric Matthews1and Oksana Lockridge2

1

Centre de Recherches du Service de Sante´ des Arme´es, Unite´ d’Enzymologie, La Tronche, France;2Eppley Institute,

University of Nebraska Medical Center, Omaha, Nebraska, USA

Butyrylcholinesterase is a serine esterase, closely related to

acetylcholinesterase Both enzymes employ a catalytic triad

mechanism for catalysis, similar to that used by serine

pro-teases such as a-chymotrypsin Enzymes of this type are

generally considered to be inactive at pH values below 5,

because the histidine member of the catalytic triad becomes

protonated We have found that butyrylcholinesterase

retains activity at pH £ 5, under conditions of excess

substrate activation This low-pH activity appears with

wild-type butyrylcholinesterase as well as with all mutants we

examined: A328G, A328I, A328F, A328Y, A328W, E197Q,

L286W, V288W and Y332A (residue A328 is at the bottom

of the active-site gorge, near the p-cation-binding site; E197

is next to the active-site serine S198; L286 and V288 form the

acyl-binding pocket; and Y332 is a component of the

peripheral anionic site) For example, the kcat value at

pH 5.0 for activity in the presence of excess substrate was

32 900 ± 4400 min)1for wild-type, 55 200 ± 1600 min)1 for A328F, and 28 700 ± 700 min)1 for A328W This activity is titratable, with pKavalues of 6.0–6.6, suggesting that the catalytic histidine is protonated at pH 5 The existence of activity when the catalytic histidine is protonated indicates that the catalytic-triad mechanism of butyrylcho-linesterase does not operate for catalysis at low pH The mechanism explaining the catalytic behaviour of butyryl-cholinesterase at low pH in the presence of excess substrate remains to be elucidated

Keywords: butyrylcholinesterase; excess substrate activation; mutant enzyme; pH dependence; steady-state kinetics

Human butyrylcholinesterase (EC 3.1.1.8; BuChE) is a

serine esterase, which is present in vertebrates It is routinely

isolated from plasma [1] where it is considered to be of

pharmacological and toxicological importance because it

hydrolyzes numerous ester-containing drugs [2] and

scav-enges toxic esters, such as organophosphates [3] Its primary

amino-acid sequence is 54% identical with that of Torpedo

californicaacetylcholinesterase (EC 3.1.1.7; AChE) [4] A

3D model for human BuChE has been built [5] from the

known co-ordinates for the 3D structure of T californica

AChE [6] This model agrees with the general features of the

recently determined X-ray structure of human BuChE [7,8]

In particular, most of the essential features of the catalytic

site (i.e a catalytic triad of Ser-His-Glu, an oxyanion hole, a

p-cation-binding site, and an acyl-binding pocket) are the

same in AChE and BuChE (Fig 1) The acyl-binding

pocket, which is responsible for the difference in substrate

specificity between the two enzymes, is larger in BuChE [5,8–10] The active site for both enzymes is located at the bottom of a 20-A˚ deep gorge An aspartate residue [D70(72)] is located at the mouth of the gorge [Italicized numbers in parentheses (N ) after amino-acid numbers refer

to residue numbering in T californica AChE In human BuChE, the corresponding residue is N)2.] This aspartate, part of the peripheral anionic site, contributes to the affinity

of positively charged substrates for the active site, and is a major factor in the binding of excess substrate to these enzymes [11,12] Neither AChE nor BuChE follows Micha-elis–Menten kinetics with positively charged substrates Under standard conditions, i.e at neutral pH and 25C, AChE has been shown to be inhibited by excess substrate, whereas BuChE is activated [13] However, we recently reported that AChE may display substrate activation at low

pH [14] The complete mechanism by which activation or inhibition of cholinesterases by excess substrate occurs is still controversial, but it is now accepted that binding of a second molecule of substrate on the peripheral anionic site (PAS) induces a conformational change that triggers the process

For some time, we have been interested in the molecular basis of substrate activation in wild-type and mutants of human BuChE [11,15,16], as well as substrate activation in wild-type and mutants of human and Bungarus fasciatus AChE at low pH [14] The term substrate activation describes the situation in which excess substrate causes an increase in the turnover number (kcat) of an enzyme For wild-type BuChE reacting with butyrylthiocholine (BTC),

Correspondence to P Masson, Centre de Recherches du Service de

Sante´ des Arme´es, Unite´ d’Enzymologie, B.P 87, 38702 La Tronche

Cedex, France Fax: + 33 4 76 63 69 63, Tel.: + 33 4 76 63 69 59;

E-mail: pymasson@compuserve.com

Abbreviations: AChE, acetylcholinesterase; BuChE,

butyrylcholine-sterase; BTC, butyrylthiocholine; DTNB,

5,5¢-dithiobis-2-nitrobenzoic acid; PAS, peripheral anionic site.

Enzymes: butyrylcholinesterase (EC 3.1.1.8; BuChE);

acetylcholin-esterase (EC 3.1.1.7; AChE).

(Received 5 August 2002, revised 29 October 2002,

accepted 25 November 2002)

the turnover number is determined by BTC concentrations

in the 10–100 micromolar range Millimolar levels of BTC

cause the activity to rise above this turnover number,

eventually reaching a new, excess-substrate-defined

turn-over number, or bkcat(with b > 1) With wild-type human

BuChE, the turnover number for BTC increases 2.5–3-fold

(b¼ 2.5–3) in the presence of excess BTC (at pH 7.0)

Mutations at the 328(330) position (A328 is at the bottom of

the active site gorge, near the p-cation site) have been

reported to cause a marked decrease in this substrate

activation, i.e A328F is activated only 20% (b¼ 1.2), at

pH 7.0 [17], whereas A328Y is inhibited by 20% (b¼ 0.8),

at pH 8.0 [9]

To clarify the cause of these differences in substrate

activation between the wild-type and A328 mutants, we

examined the pH dependence of BTC turnover Although

we found the pKavalues for kcatand bkcatof the mutants to

be slightly decreased relative to wild-type, these decreases

could not explain the observed differences in substrate

activation However, in the course of our studies, we

observed that the bkcatvalues did not fall to zero as the pH

was lowered from 8.5 to 5.0, for either wild-type or mutant

BuChE Rather they approached a substantial, nonzero

limit For example, the limiting value for bkcatat low pH

was 32 900 ± 4400 min)1 for wild-type, 55 200 ±

1600 min)1 for A328F, and 28 700 ± 700 min)1 for

A328W Similar observations were made for wild-type

human and B fasciatus AChEs and their mutants modified

on the equivalent residues [14] Consistent with previous

reports, we found that kcat did approach zero as the pH

decreased from 8.5 to 5.0 The pKavalues that we found for

both kcat and bkcat are consistent with titration of the

catalytic histidine, H438(440) The persistence of activity in

the presence of the protonated form of the catalytic histidine

is inconsistent with the generally accepted mechanism for

hydrolysis by cholinesterases [18,19] This mechanism

utilizes the catalytic histidine as an acceptor for a proton from the catalytic serine, therefore protonation of the histidine would be expected to block catalysis Rather, the appearance of activity under conditions in which the catalytic histidine is protonated indicates a change in the mechanism of BuChE and AChE Work is in progress to probe the mechanism that could explain these observations

Materials and Methods

Chemicals Butyrylthiocholine iodide (BTC) and 5,5¢-dithiobis-2-nitro-benzoic acid (DTNB) were purchased from Sigma Chemical Co., St Louis, MO, USA Chlorpyrifos-oxon was from Chem Services Inc., West Chester, PA, USA (catalog number MET-674B) All other chemicals, including buffer components, were of biochemical grade

Mutagenesis and expression of recombinant BuChE Mutagenesis and expression were performed as described previously [17] Briefly, mutations in human BuChE were created, then amplified by PCR using Pfu polymerase Fragments containing the mutation were cloned into the plasmid pGS The plasmid was transfected into CHO-KI cells by calcium phosphate coprecipitation Stable cell lines were selected in methionine sulfoximine Expressed BuChE was secreted from these cells and collected into serum-free medium

Purification of BuChE Mutant forms of BuChE were purified from culture medium as previously described [1,17] Briefly, the culture medium was passed over a procainamide–Sepharose affinity column which retained the BuChE, which was then selectively eluted with 0.2M procainamide hydrochloride Further purification was obtained by ion-exchange chro-matography on DE52 (Whatman, Clifton, NJ, USA) using

an NaCl gradient for elution The resulting enzyme was typically 70–95% pure Wild-type BuChE used in this work was purified from human plasma using the same combina-tion of affinity and DE52 chromatography

The concentration of each BuChE mutant was deter-mined by titration with chlorpyrifos-oxon as proposed by Amitai et al [20] Chlorpyrifos-oxon concentration was standardized against wild-type BuChE of known concen-tration

Enzyme assay Initial rate of turnover of BTC was measured by the method

of Ellman et al [21] in 0.1Msodium phosphate buffer, pH variable from 5.0 to 8.5 and, in 0.1Msodium acetate buffer,

pH ranging from 4.0 to 5.25 The ionic strength of phosphate buffers varied from 0.1 to 0.29, and that of acetate buffers varied from 0.014 to 0.075 Such changes in ionic strength are known to have no effect on kcat of BuChE-catalyzed hydrolysis of cationic substrates [11,13,22] Buffers contained 0.33 mM DTNB and 0.01–

50 m BTC, at 25C Product formation was followed by

Fig 1 Side view of the active-site gorge of acylated (butyrylated) human

BuChE The arrow indicates the entrance of the gorge D70 and Y332

are the peripheral anionic site residues The active site is at the bottom

of the gorge: the substrate binding subsite is W84 and A328; the

acyl-binding pocket is formed from L286 and V288; the catalytic triad is

S198, H438 and E325 Residue 197 next to the catalytic serine is

involved in stabilization of transition states.

the change in A420 Observed rates were corrected for

spontaneous hydrolysis of BTC and for spontaneous

reduction of DTNB from blank samples The observed

rate, in terms of mol productÆmin)1ÆL)1, was obtained by

dividing the DA420Æmin)1by the absorption coefficient for

the 5-thio-2-nitrobenzoic acid (TNB), which is the product

of the reaction of thiocholine with DTNB This calculation

was complicated by the fact that TNB has a pKaof 4.53 [23],

which gives rise to a decrease in absorption coefficient at pH

values below 7.0 Consequently, we measured the

absorp-tion coefficients at 420 nm for TNB, at pH values between

5.0 and 8.5 Representative absorption coefficients are:

8590M )1Æcm)1 at pH 5.0; 11 100M )1Æcm)1 at pH 5.5;

12 500M )1Æcm)1 at pH 6.0; 13 200M )1Æcm)1 at pH 7.0;

and 13 300M )1Æcm)1at pH 8.0

Data analysis

Steady-state turnover of BTC with wild-type BuChE

exhibits the phenomenon of excess substrate activation

This is illustrated in Scheme 1 This scheme is also suitable

for excess substrate inhibition

This scheme is described by Eqn (1):

kapp¼ kcatþ

bk cat þ½S

Kss

1þK m

½S

1þK½S

ss

where kapp is the apparent rate, in terms of mol

productÆ(mol BuChE))1Æmin)1, [S] is the concentration of

BTC kcat is the turnover number (min)1) when

[S] << Kss, Km is the Michaelis–Menten constant, bkcat

is the turnover number (min)1) when [S] >> Kss, and Kss

is the dissociation constant for excess BTC [10,24] The

parameter b reflects the efficiency of product formation

from the ternary complex (SES) When b > 1, there is

substrate activation When b < 1, there is substrate

inhibition When b¼ 1, the enzyme follows Michaelis–

Menten kinetics The kcat, Km, Kss and b values were

obtained by nonlinear fitting of the apparent rate vs BTC

concentration data to Eqn (1), using SigmaPlot v4.16

(Jandel Scientific, San Rafael, CA, USA) The value for

bkcatwas obtained by multiplying kcatby b

Results

pH dependence of turnover

The values of kcat, bkcat, Kmand Ksswere obtained by fitting

the apparent rate (kapp) vs BTC concentration data to eqn

(1), at each pH value (data not shown) To within the limits

of experimental error, Kmwas independent of pH for all

enzymes This is consistent with earlier reports on the pH

dependence of K for human BuChE [25,26]

The value of Kssincreased as the pH was lowered from 8.5 to 5.2, for all enzymes This indicates that the binding of BTC to the excess-substrate activation site is becoming weaker as the pH is lowered The change in Kssvaried from fivefold to 20-fold, depending on the enzyme At pH 8.5, Kss had essentially stopped changing with pH, having reached a limiting value for high pH As the pH was lowered, the value

of Kssbecame progressively larger; however, by pH 5.2 a clear inflection point had not yet developed Therefore, pKa values for Ksscould not be determined; only an upper limit

of 5.0 could be estimated Such a low pKais consistent with the involvement of an acidic amino acid in the binding of excess BTC Excess-substrate activation for human BuChE has been attributed to binding of positively charged substrates, such as BTC, to D70 in the peripheral anionic site [11,27] The pH dependence of Kssis consistent with protonation of D70 The pH dependence data for the D70G mutant supports this statement Indeed, although the D70G mutant shows a slight activation by excess substrate (b¼ 1.2 ± 0.2) at BTC concentrations higher than 2 mM [11,15,27], its high Kss value (> 1 mM [27]) does not significantly change with pH (not shown) However, D70 is not the only residue involved in substrate activation: the D70N mutant was shown to be strongly activated by excess substrate [27] and even the D70G mutant shows substrate activation similar to that of wild-type enzyme in the presence of high concentrations of sugars or polyols [28]

No further discussion on the pH dependence of Kmor Kss will be presented, so that we can focus attention on the pH dependence of kcatand bkcat

Figure 2 shows the pH dependence of kcatand bkcatfor wild-type BuChE and the mutants A328G, A328I, A328F, A328Y and A328W, using BTC as substrate Simple inspection of Fig 2 reveals that both kcatand bkcatexhibit well-defined pH titration profiles, with the minimum rates occurring at low pH The values for kcatapproach zero by

pH 5.0 However, the bkcat values clearly approach a nonzero limiting activity at low pH Limiting activity is the plateau value in a titration curve All of the titrations extend over a range of at least 3 pH units, indicating that they are more than 90% complete by pH 5 Therefore, the nonzero limiting rates for bkcatat low pH cannot be attributed to incomplete titration

The data in Fig 2 were in turn fitted to an expression for

a single pKa (see the legend to Table 1 for details) The fitting results are tabulated in Table 1

Wild-type BuChE and all of the A328 mutants show limiting rates for kcatat low pH (kH) that are 10% or less of their limiting rates at high pH (kA) Therefore, the limiting values of kcatat low pH may be considered to be effectively zero This is despite the fact that most of these kHvalues are statistically greater than zero The pKavalues of 6.5–7.0 for these enzymes are all consistent with the titration of a histidine These results for kcatare consistent with the what

is commonly found for wild-type BuChE and wild-type AChE (see [32] for a review) In these reports, the titrating histidine has consistently been taken to be the catalytic histidine, i.e H438(440) in BuChE

The bkcat values also gave well-behaved titrations for wild-type BuChE and all of the A328 mutants, except A328W The pKa values of 6.0–6.5 are consistent with titration of a histidine By analogy with k , the titrating Scheme 1 Steady state turnover of BTC.

histidine is probably the catalytic histidine, H438 It is

noteworthy that the limiting values of bkcatat low pH are

decidedly greater than zero (e.g kH¼ 32 900 ± 4400 min)1

for wild-type BuChE or kH¼ 28 700 ± 700 min)1 for

A328W) Thus, in the presence of excess BTC, BuChE is

active even though H438 is protonated The existence of

substantial activity for BuChE when the catalytic histidine is

protonated is an unprecedented observation, which has

significant implications for the mechanism

We would like to emphasize that the activity that we

measure for bkcatdoes not approach zero at low pH For a

titration that ends at zero activity for the fully protonated

histidine, theory predicts that at 1 pH unit below the

pKa, only 10% of the histidine is unprotonated and that

therefore only 10% of the activity will remain Our results

in Fig 2 show that for A328F 71% remains, for A328I

32% remains, for wild-type 40% remains, and for A328G

42% remains In all four cases, the remaining activity at

1 pH unit below the pKais much higher than the theoretical

prediction

As the major point of this paper rests on the observation

that there is significant turnover of BTC by BuChE at low

pH, it is important to control for artifactual sources of

turnover The first point to be made is that, to our

knowledge, BTC is not hydrolyzed, to any significant extent,

by any enzyme other than BuChE AChE will hydrolyze

BTC slowly, but our recombinant enzymes were collected into serum-free medium, which contains no AChE As we are seeing high bkcatvalues for BTC turnover at low pH, this activity is probably not due to a contaminating enzyme Secondly, all of the rate data have been corrected for spontaneous hydrolysis of BTC and chemical reduction of DTNB Thirdly, to control for unexpected contaminations

in the expressed enzymes, which might have arisen from the cell culture, we examined wild-type BuChE that had been purified to homogeneity (tetrameric G4form) from human plasma As can be seen in Figs 2 and 3, wild-type BuChE also had substantial activity at low pH, in the presence of excess substrate It is unlikely that both naturally occurring and cultured BuChE would show the same contaminations; therefore, artifactual hydrolysis from contamination is unlikely

Further titration of wild-type BuChE The titration of wild-type BuChE was extended from

pH 8.5 to 4.0 At pH 4.0, the kcatactivity was effectively zero, and bkcatactivity was approaching zero (Fig 3) The titration of kcatwas monophasic, with a pKaof 6.7 ± 0.09 However, the titration of bkcatwas very broad, extending for more than 4.5 pH units The profile was clearly biphasic, with pKavalues of 4.63 ± 0.24 and 6.68 ± 0.20 and a rate for the singly protonated species of 41 400 ± 4500 min)1 Complete elimination of the bkcatactivity required proto-nation of two amino acids This biphasic titration accen-tuates the fact that the intermediate, singly protonated species is active That is to say, complete protonation of

an amino acid with a pKaof 6.68 results in an enzyme that still hydrolyzes BTC at a rate of 41 400 min)1 As we have suggested, the most likely candidate for this group is the catalytic histidine, H438 The pKa of 4.63 suggests the involvement of an acidic residue in the activity at low pH

Low-pH activity and residue E197 Selwood et al [34] reported two pKa values for the pH dependence of the reaction of Electrophorus electricus and

T californicaAChEs with BTC The higher pKa(6.3 and 6.1, respectively) was attributed to the titration of the catalytic histidine, H440 (corresponding to H438 in human BuChE) Protonation of this residue in electric eel AChE left

a preparation that retained 30% of the activity found at high pH The residual activity could be abolished by further titration, yielding a pKaof 4.7 Similarly, the pH profile for

T californica AChE yielded a pKa of 5.0 This pKa was attributed to residue E199, an active-site residue corres-ponding to E197 in human BuChE They proposed that titration of H440 resulted in a change in mechanism from triad catalysis to one that likely involves general base catalysis by E199 of direct water attack on the scissyl carbonyl The similarity between their results and ours is evident and suggests that the low-pH activity of BuChE, in the presence of excess BTC, may be due to general base catalysis by E197

Owing to this similarity, it became necessary to test the involvement of residue E197(199) in the activity of BuChE

at low pH To accomplish this, we determined the pH

Fig 2 pH dependence for the turnover number (k cat ) and the

excess-substrate-activated turnover number (bk cat ) of wild-type human BuChE

and various 328-position mutants Each panel represents a different

mutant form of BuChE, as indicated In each panel, the solid circles

indicate the measured k cat values, the solid squares indicate the

measured bk cat values, and the lines are the result of fitting the

meas-ured rates to an expression for a single pK a (see the legend to Table 1

for details) The values for k cat and bk cat , at each pH, were taken from

fitting of k app vs BTC concentration data for each mutant (data not

shown).

dependence of the activity of the BuChE mutant E197Q.

We reasoned that glutamine at position 197 was

equiv-alent to protonation of E197 If general base catalysis by

E197 was responsible for the residual activity seen with

wild-type BuChE, in the presence of excess BTC at low

pH, then E197Q would not be able to support that

activity The excess-substrate activity of E197Q (bkcat)

should then titrate to zero with a pKa 6.5

(correspond-ing to the catalytic histidine, H438) Figure 4 shows that

this expectation was not realized The bkcatrates of E197Q

approach a substantial, limiting rate at low pH The

change in bkcatbetween high pH and low pH is not large,

but the trend is clear, and it yields a pKaof 6.17 ± 0.56

Thus, the suggestion that E197 is responsible for the

low-pH activity of wild-type BuChE in the presence of excess

BTC is not supported

Moreover, it should be noted that the pKa of mutant

E1997Q for kcatis shifted by 1 pH unit below that of kcat

of wild-type Such a shift supports the assumption that the

observed pKais related to His438 because it is consistent

with the fact that the electrostatic stabilizing effect of the

E197 side chain on the protonated form of H438 is

abolished in the E197Q mutant

Role of position 328 in the excess-substrate effect

Mutations at position 328 seem to modulate the behaviour

of BuChE, rather than to introduce qualitatively new

behaviour The most obvious indication of this modulation

appears at high pH where the limiting value of bkcattends to

approach the limiting value of kcatas the size of the residue

at position 328 increases (Fig 2) For example, the

differ-ence between bkcatand kcatat high pH is 68 400 min)1for wild-type BuChE (A328), 19 700 min)1 for A328F, and

13 000 min)1for A328W Thus, the larger residues seem to interfere with the ability of excess substrate to increase the activity at high pH However, the aliphatic or aromatic character of side chains has to be considered too At low

pH, the size of the residue in position 328 has no consistent effect on bkcat The values for bkcatare generally between

20 000 and 40 000 min)1(Fig 2) The net effect is that the value of bkcatat high pH becomes closer to its value at low

pH as the size of the residue at position 328 gets larger (Fig 2) For example, the difference between bkcatat high

pH and bkcat at low pH is 65 600 min)1 for wild-type (A238), 22 700 min)1for A328F, and 0 for A328W From the effect that the size of the 328 residue has on

bkcat, it is tempting to suggest that the 328 position (which is part of the substrate binding site) plays a special role in the excess substrate effect However, mutations at other loca-tions in the active site also perturb the pH dependence of

bkcat E197Q (part of the esteratic site) shows a pH dependence for bkcat that is similar to that for A328F (Fig 4) V288W(in the acyl-binding pocket) and Y332A (in the PAS) show pH dependencies more like A328I, i.e the difference between bkcatand kcatat high pH is smaller than for wild-type, and the difference between bkcatat high pH and bkcatat low pH is relatively small (Fig 5) On the other hand, L286W(also in the acyl-binding pocket) is similar to wild-type BuChE, i.e the difference between bkcatand kcat

at high pH is large relative to the difference at low pH, and the difference between bkcatat high pH and bkcatat low pH

is relatively large (Fig 5) In all of these enzymes, there is a substantial activity for bk at low pH, strengthening the

Table 1 pH dependence of k cat and bk cat for BuChE mutants Values for the parameters were determined by fitting the data from Figs 1, 3 and 4 to the expression:

k ¼kHþ kA 10

ðpH pK a Þ

1 þ 10 pH pK a

which is an algebraic rearrangement of the more common expression for the dependence of rate on pH involving a single pK a [29]:

pH ¼ pK a log k kA

k H k

The term k stands for the observed rate, k A stands for the limiting rate at high pH, and k H stands for the limiting rate at low pH This is a general expression for a pH titration, which does not exclude the possibility of a nonzero limiting rate at either pH extreme The rearrangement was required in order to obtain the dependent variable (k) in terms of the independent variable (pH) Fitting was performed using SIGMAPLOT v.4.16 kcatis the turnover number in the absence of excess substrate bkcatis the turnover number in the presence of excess substrate Residue volumes were taken from Zamyatnin [30] Hydrophobicity ratings were taken from Karplus’ pure hydrophobicity scale [31] NA, not applicable There is no change in bk cat with pH for A328W.

BuChE

Residue volume (A˚ 3 ) Hydrophobicity

kH

(min)1)

kA (min)1) pKa

kH (min)1)

kH (min)1) pKa A328G 2000 ± 1100 26 100 ± 1000 6.78 ± 0.11 26 800 ± 1700 70 400 ± 800 6.23 ± 0.07 60.1 1.18

Wild-type 2800 ± 1100 30 100 ± 1200 6.83 ± 0.10 32 900 ± 4400 98 500 ± 3600 6.56 ± 0.15 88.6 2.15

A328I 1230 ± 1100 27 900 ± 1340 6.79 ± 0.11 13 600 ± 3640 51 100 ± 1480 6.02 ± 0.15 166.7 3.88

A328F 6600 ± 1300 58 200 ± 1800 6.58 ± 0.06 55 200 ± 1600 77 900 ± 2200 6.57 ± 0.18 189.9 3.46

A328Y 8200 ± 3000 73 300 ± 3100 7.03 ± 0.11 33 400 ± 9400 71 900 ± 2600 6.12 ± 0.32 193.6 2.81

A328W3200 ± 1900 41 000 ± 1700 6.59 ± 0.13 28 700 ± 700 28 700 ± 700 NA 227.8 4.11

E197Q 3200 ± 740 12 000 ± 280 5.85 ± 0.12 12 800 ± 1200 16 700 ± 730 6.17 ± 0.56 – –

L286W4300 ± 2700 16 200 ± 2100 6.26 ± 0.45 17 400 ± 4100 93 000 ± 2700 6.17 ± 0.10 – –

V288W6300 ± 1100 52 200 ± 1800 6.70 ± 0.07 62 900 ± 4200 89 300 ± 4200 6.08 ± 0.29 – –

Y332A 2900 ± 1400 48 400 ± 1400 6.51 ± 0.07 27 500 ± 3400 64 700 ± 2600 6.29 ± 0.19 – –

argument that this high activity at low pH in the presence of

excess substrate is a common feature of BTC hydrolysis by

BuChE

Mutations in the acyl-binding pocket of mouse AChE

(F297A and F297I) or in the hydrogen-bonding network

(E450Q) have also been shown to alter the excess-substrate effect In these cases, excess-substrate inhibition was switched into excess-substrate activation [10,35,36] More-over, mutations in the p-cation-binding site of human and snake AChE have been found to cause activation by excess acetylthiocholine at low pH [14] Taken together, these observations suggest that any change to the structure of the active site may alter the excess-substrate effect Thus the response of cholinesterases to excess-substrate binding appears to involve the intricate interplay of a variety of residues in the active site

pH dependence of A328W The A328Wmutant has a remarkable pH vs activity profile (Fig 2, bottom right panel) At low pH, bkcatis larger than

kcat, but at high pH bkcatis smaller than kcat That is to say, A328Wgoes from substrate activation, at low pH, to substrate inhibition, at high pH A similar observation was made by Kalow [25] using benzoylcholine as substrate for wild-type human BuChE and by us with benzoylthiocholine

as substrate on the same enzyme In particular, the pH-dependence study of benzoylthiocholine hydrolysis by wild-type BuChE showed a progressive shift from activation by excess substrate (b > 1) at low pH to inhibition by excess substrate (b < 1) at pH > 7.1 (unpublished)

There is no reason to believe that excess substrate binds to

a different site at high pH than it does at low pH Therefore the switch from substrate activation to substrate inhibition most probably reflects a pH-dependent difference in the response of the protein to excess-substrate binding That is

to say, the structure of the BuChE active site changes in response to excess-substrate binding, and this change is different at high pH than it is at low pH

Fig 4 pH dependence for the turnover number (k cat ) in the absence of

excess substrate and for the turnover number (bk cat ) in the presence of

excess substrate, of the human BuChE mutant E197Q The solid circles

indicate the measured k cat values, the solid squares indicate the

measured bk cat values, and the lines are the result of fitting the

meas-ured rates to an expression for a single pK a (see the legend to Table 1

for details) The values for k cat and bk cat , at each pH, were taken from

fittings of k vs BTC concentration data (data not shown).

Fig 5 pH dependence for the turnover number (k cat ) in the absence of excess substrate and for the turnover number (bk cat ) in the presence of excess substrate, of various BuChE mutants Each panel represents a different mutant form of BuChE, as indicated In each panel, the solid circles indicate the measured k cat values, the solid squares indicate the measured bk cat values, and the lines are the result of fitting the meas-ured rates to an expression for a single pK a (see the legend to Table 1 for details) The values for k cat and bk cat , at each pH, were taken from fittings of k app vs BTC concentration data (see Materials and Meth-ods) for each mutant (data not shown).

Fig 3 pH dependence for the turnover number (k cat ) in the absence of

excess substrate and for the turnover number (bk cat ) in the presence of

excess substrate, of wild-type human BuChE over the pH range 4–8.5.

From pH 5 to 8.5 the assays were performed in 0.1 M sodium

phos-phate buffers From pH 4 to 4.75 the assays were performed in 0.1 M

sodium acetate buffers The solid circles indicate the measured k cat

values, the solid squares indicate the measured bk cat values, and the

lines are the result of fittings The k cat rates were fitted to an expression

for a single pK a (see the legend to Table 1 for details) The bk cat rates

were fitted to an expression for two pK a values [33].

k ¼kA K2 K4þ kH ½H  K4þ kH2 ½H

2

K 2  K 4 þ ½H  K 4 þ ½H 2

The term k is the observed rate, k A is the limiting rate at high pH, k H is

the rate for singly protonated form, k H2 is the limiting rate at low pH

(zero in this case), K 2 is the dissociation constant for the first

proto-nation, K 4 is the dissociation constant for the second protonation, and

[H] is the hydrogen ion concentration The values for k cat and bk cat , at

each pH, were taken from fittings of k app vs BTC concentration data

(data not shown).

Dependence of pKaon the size/hydrophobicity

or aromaticity of residue 328

The original motivation for these studies was the

hypo-thesis that the residue in position 328 significantly

perturbed the pKavalues for kcatand bkcat This, however,

is not the case There is only a slight dependence of pKa

on the size of the residue at position 328 (Fig 6) It is,

however, noteworthy, that with any given mutant the pKa

for bkcatis generally lower than the pKafor kcat(Table 1)

This difference indicates that excess-substrate binding to

the PAS site of BuChE affects the environment of H438

It could be due to either the presumed conformational

change induced on binding [11,12,15,37,38] or simply the

presence of an additional positive charge close to the

active site (in the form of BTC or any positively charged

substrate) making protonation of the catalytic histidine

more difficult

Plots of pKavalues against the nonpolar surface area of

the residue [31] or against any of a variety of residue

hydrophobicity scales, e.g Chothia’s residue accessible

surface area scale [39] or Nozaki and Tanford’s water/

organic solvent partition scale [40], were similar to those in

Fig 6 (data not shown) It is not surprising that the

correlation of pKavalues with residue size is similar to the

correlation of pKa values with residue hydrophobicity, as

Chothia [41] has pointed out that hydrophobicity is

directly related to the accessible surface area of the

residue, i.e size In view of this, we believe that it is not

possible to conclude whether the variations in pKaof bkcat

of the A328 mutants are due to a steric or a

hydropho-bic effect Moreover, results with the bulkiest residue

(mutant A328W) do not fit the pattern, suggesting

that the tryptophan ring may affect the H438 pKathrough

p-cation interactions

Discussion

The central observation in this paper is that BuChE retains

significant hydrolytic activity after protonation of what

appears to be the catalytic histidine, H438 This occurs under the influence of binding of excess substrate to the PAS, i.e for bkcat, but not at lower substrate concentrations, i.e for kcatin Scheme 1 This creates a major mechanistic puzzle

How can BuChE manage to turnover at a rate of

 20 000–50 000 min)1when the catalytic histidine appears to be protonated?

Let us review the accepted general mechanism of serine hydrolase catalysis The mechanism of serine esterases is generally considered to be analogous to that of the serine proteases [18] It goes through four steps [18,42,43] represented in Scheme 2 (human BuChE amino-acid numbering) First, the carbonyl carbon of the substrate undergoes a nucleophile attack by the Oc of the catalytic serine, while the proton is shuttled to the catalytic histidine This results in the formation of a tetrahedral transition-state intermediate, the negative charge on the former carbonyl oxygen being stabilized by interactions with the dipoles of the oxyanion hole Secondly, the alcohol product

is released, picking up a proton from the catalytic histidine This results in the formation of a transient acyl-enzyme adduct The alcohol product is exchanged with a molecule

of water Thirdly, the acyl-enzyme undergoes a nucleophile

Scheme 2 Proton shuttle mechanism.

Fig 6 Dependence of pK a for k cat (filled circles) and bk cat (open

squares) on the volume of the residue at position 328 The pK a values

were taken from fitting the data of Fig 1 to an expression for a single

pK a (see Table 1) The residue volume was taken from Zamyatnin [30]

(see Table 1) The letters are the single letter codes for the amino acids

at the 328 position They are provided to help the reader to associate

the data with the mutant The lines are presented to emphasize the

trend in these data They have no analytical significance.

attack by this molecule and the dissociated proton is

transferred to the catalytic histidine This results in the

formation of a second tetrahedral transition-state

interme-diate, the negative charge on the former carbonyl oxygen

being again stabilized by the oxyanion hole Fourthly, the

catalytic serine is released, picking up a proton from

the catalytic histidine This results in regeneration of the

starting enzyme

There are currently two proposals for the driving force

behind catalysis: the low-barrier hydrogen-bond model

[44,45] and the electrostatic stabilization of the transition

state model [46,47]

According to the low-barrier energy model, substrate

binding drives a conformational change to form a

Michaelis complex in which steric compression is

intro-duced between the histidine and carboxylate (aspartate in

chymotrypsin, glutamate in cholinesterases) of the

cata-lytic triad Compression of the His-Asp/Glu diad causes

the basicity of the histidine to increase, so that it is able to

accept/abstract the Oc proton from the catalytic serine

Transferring a proton from the catalytic serine to the

catalytic histidine relieves the steric compression by

forming a short, strong hydrogen bond between the

protonated histidine and the carboxylate In this way, the

ability of the histidine to accept the proton from the serine

Oc is greatly increased The presence of a short, strong

hydrogen bond was shown by NMR studies for both

human AChE and human BuChE complexes with

com-pounds mimicking the transition state [48,49]

In the electrostatic transition-state stabilization model,

the dipoles of the oxyanion hole are considered to be

optimally prealigned to strongly polarize the carbonyl of the

substrate The carbonyl carbon becomes very electrophilic

and as the bond between the serine c-oxygen and the

carbonyl carbon forms, the pKaof the Oc proton falls to a

point where it can be released to the catalytic histidine,

which is positioned to accept it Thus, no free energy is spent

on re-orienting the dipoles of the protein in the transition

state This leads to a large decrease in transition-state energy

for the enzyme reaction compared with the chemical

hydrolysis reaction in water, and thereby, a large increase

in the rate for catalysis It is noteworthy that the ability of

the oxyanion hole to stabilize the tetrahedral transition state

is well illustrated in the recently solved X-ray structure of

human BuChE [8] In this structure, the enzyme is not free,

but the acidic product of substrate hydrolysis (butyrate) is

still loosely bound to the catalytic serine (bond length is

2.16 A˚) The carbonyl carbon of the butyrate adopts a

partial tetrahedral character Such a distortion results from

the strong polarization of the C–O bond by the dipoles of

the oxyanion hole in conjunction with the influence of a

close nucleophile like the Oc of the catalytic serine The

same type of adduct was observed previously for

Strepto-myces griseusprotease A [50] Thus, the butyrate–BuChE,

quasi-tetrahedral complex is more stable than the free or the

nonhydrated acyl-enzyme

We have found that the protonated form of BuChE, in

the presence of excess substrate, i.e bkcat, is active at low pH

(see Fig 2) It is assumed that the change in bkcat as a

function of pH reflects protonation of the catalytic histidine

This observation generates a problem for any mechanism in

which the catalytic histidine is the proton acceptor for the

catalytic serine, because the likelihood of a protonated histidine accepting an additional proton is very low Thus,

at low pH and in the presence of excess substrate, any model for catalysis by human BuChE based on the histidine being the proton acceptor becomes untenable The protonated form of the catalytic histidine may accept a proton in the transition state, therefore serving as a general base catalyst, only if a concerted proton transfer to the leaving group of the substrate occurs

Proton transfer The requirement of an acceptor for the Oc proton from the catalytic serine is a critical component of the mechanism for the serine proteases/esterases Based on the 3D structures of a-chymotrypsin [44], AChE [6], and human BuChE [8], the most logical recipient for the serine proton is the catalytic histidine Most models use the histidine to shuttle protons: first, between the serine and the leaving group; and then between the attacking water and the serine In its role as a proton shuttle, the catalytic histidine formally accepts a proton from a donor and then delivers it to the acceptor (Scheme 3)

On the other hand, our data at low pH indicate that catalysis by BuChE and AChE [14], in the presence of excess substrate, can proceed readily even when the catalytic histidine is protonated Formal transfer of an additional proton to the cationic form of the histidine is not reasonable This is the dilemma One solution would be a concerted proton transfer, in the transition state, via the catalytic histidine The concerted proton transfer would be between the serine and the leaving group and later between the water and the serine

Cyclic transition-state structures have been proposed for nonenzymatic acid catalysis of ester hydrolysis ([51] and references therein) Such a concerted transition state would not conflict with electrostatic stabilization of the transition state Finally, a concerted proton transfer mechanism could also explain catalysis by AChE at low pH [14] How-ever, further studies are needed to test this hypothetical mechanism

Thus, we suggest that BuChE and AChE may use two different mechanisms for transferring protons At high pH, where the catalytic histidine is unprotonated, both choli-nesterases use the traditional proton shuttle mechanism (Scheme 3) both for kcatand bkcat At low pH (where the catalytic histidine is protonated), and in the presence of excess substrate, the binding of which induces a conform-ational change, cholinesterases use another mechanism which remains to be elucidated

Scheme 3 Proton shuttle transition state.

We have found that, as for AChE [14,34], the turnover of

human BuChE reaches a substantial, nonzero limiting rate

at low pH, in the presence of excess positively charged

substrate This observation suggests that catalysis at low

pH, in the presence of excess substrate, does not involve the

classical acid-base triad-mediated mechanism However,

involvement of general base catalysis by a carboxylate, i.e

E197(199), was disproved

The observations of high activity from BuChE and

AChE at low pH is a new and important finding which

requires further investigation to dissect the molecular

mechanisms of hydrolysis of substrates by cholinesterases

under extremes of pH and substrate concentration

Acknowledgements

We are grateful to Lawrence M Schopfer for fruitful discussions and

constant support during this work This work was supported by US

Medical Research and Materiel Command grants DAMD

35-1905-2010-00 (to O.L.), DGA/DSP/STTC 99CO-029/PEA and DGA/

ODCA Washington 00-2-032-0-00 (to P.M and O.L.) and by a

National Cancer Institute grant, P30CA36727, to the Eppley

Institute The opinions and assertions contained herein should not

be construed as the official views of the US Army or the Department

of Defense.

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