Hydrolysis of oxo- and thio-esters by human butyrylcholinesterase

Biochimica et Biophysica Acta 1774 (2007) 16 – 34 www.elsevier.com/locate/bbapap

Hydrolysis of oxo- and thio-esters by human butyrylcholinesterase Patrick Masson a,⁎, Marie-Thérèse Froment a , Emilie Gillon a , Florian Nachon a , Oksana Lockridge b , Lawrence M. Schopfer b a

Centre de Recherches du Service de Santé des Armées, Unité d’Enzymologie, BP 87, 38702 La Tronche Cedex, France b University of Nebraska Medical Center, Eppley Institute, 600 S 42nd St., Omaha, NE 68198-6805, USA Received 7 January 2006; received in revised form 25 October 2006; accepted 26 October 2006 Available online 9 November 2006

Abstract Catalytic parameters of human butyrylcholinesterase (BuChE) for hydrolysis of homologous pairs of oxo-esters and thio-esters were compared. Substrates were positively charged (benzoylcholine versus benzoylthiocholine) and neutral (phenylacetate versus phenylthioacetate). In addition to wild-type BuChE, enzymes containing mutations were used. Single mutants at positions: G117, a key residue in the oxyanion hole, and D70, the main component of the peripheral anionic site were tested. Double mutants containing G117H and mutations on residues of the oxyanion hole (G115, A199), or the π-cation binding site (W82), or residue E197 that is involved in stabilization of tetrahedral intermediates were also studied. A mathematical analysis was used to compare data for BuChE-catalyzed hydrolysis of various pairs of oxoesters and thio-esters and to determine the rate-limiting step of catalysis for each substrate. The interest and limitation of this method is discussed. Molecular docking was used to analyze how the mutations could have altered the binding of the oxo-ester or the thio-ester. Results indicate that substitution of the ethereal oxygen for sulfur in substrates may alter the adjustment of substrate in the active site and stabilization of the transition-state for acylation. This affects the k2/k3 ratio and, in turn, controls the rate-limiting step of the hydrolytic reaction. Stabilization of the transition state is modulated both by the alcohol and acyl moieties of substrate. Interaction of these groups with the ethereal hetero-atom can have a neutral, an additive or an antagonistic effect on transition state stabilization, depending on their molecular structure, size and enantiomeric configuration. © 2006 Elsevier B.V. All rights reserved. Keywords: Butyrylcholinesterase; Ester; Thio-ester; Oxyanion hole; Mutation; Rate-limiting step

1. Introduction Butyrylcholinesterase (BuChE; EC 3.1.1.8) and acetylcholinesterase (AChE; EC 3.1.1.7) are closely related serine esterases [1]. Both enzymes hydrolyze choline esters and other esters. Due to a larger substrate-binding pocket that can accommodate bulky substrates [2], the specificity of BuChE is Abbreviations: AChE, Acetylcholinesterase; ACh, acetylcholine; ASCh, acetylthiocholine; BuChE, butyrylcholinesterase; BuCh, butyrylcholine; BuSCh, butyrylthiocholine; BzCh, benzoylcholine; BzSCh, benzoylthiocholine; CPO, chlorpyrifos-oxon; DFP, diisopropylfluorophosphate; OP, organophosphate; PAS, peripheral anionic site; PhA, phenylacetate; PhSA, phenylthioacetate; PrCh, propionylcholine; PrSCh, propionylthiocholine; SdCh, succinyldicholine; SdSCh, succinyldithiocholine ⁎ Corresponding author. Fax: +33 4 76 63 69 62. E-mail addresses: [email protected], [email protected] (P. Masson). 1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2006.10.012

much larger than that of AChE. Unlike AChE which plays a key role in cholinergic synapses in terminating the action of the neurotransmitter acetylcholine, no clear physiological function (s) has(ve) yet been assigned to BuChE. However, there is increasing evidence for the involvement of this enzyme in noncholinergic functions such as cell differentiation, neurogenesis and formation of amyloid plaques in Alzheimer's disease [3–5]. AChE knock-out mouse studies indicate that BuChE in the central nervous system could have a surrogate acetylcholinehydrolyzing function [6]. Because BuChE is capable of hydrolyzing numerous natural and artificial esters it is also an enzyme of pharmacological and toxicological importance [7– 10]. Moreover, like AChE, BuChE is inhibited by organophosphoryl- and carbamyl-esters through phosphylation or carbamylation of its active site serine [11]. Certain of these toxic esters are pesticides and potent nerve agents [12,13], others are current or promising anti-Alzheimer drugs [14].

P. Masson et al. / Biochimica et Biophysica Acta 1774 (2007) 16–34

Scheme 1.

Natural or recombinant wild-type human BuChE is a stoichiometric scavenger of OP. It is currently the leading candidate for detoxification of OP poisoning, and is being considered for use in prophylaxis and treatment of OP poisoning, particularly for poisoning by chemical warfare nerve agents [15,16]. Mutants of human BuChE have been designed to hydrolyze OP for use as catalytic scavengers [17]. Though these mutants are still too slow to be of operational interest, it should be noted that a transgenic mouse expressing G117H human BuChE is resistant to OP poisoning [18]. The catalytic properties of cholinesterases have been extensively investigated (for reviews see ref. [19,20]). The hydrolysis of substrates by AChE and BuChE can be described by the Michaelis–Menten model (Scheme 1). However, this simple model is valid only for neutral substrates, or for positively charged substrates at low concentration: where Ks = (k− 1 + k2) / k1 is the dissociation constant the enzyme–substrate complex, k2 the rate constant of acylation, and k3 the rate constant of deacylation. P1 is the alcohol/phenol hydrolysis product and P2 the carboxylic acid product. m¼

kcat d½E½S Km þ ½S

kcat ¼ Vmax =½ E  ¼

ð1Þ k2 k3 ðk2 þ k3 Þ

ð2Þ

Eq. (2) can be re-written as kcat ¼ 

k2 1þ

k2 k3

 or 

k3 1 þ kk32



ð2bis Þ

and Km ¼

ðk1 þ k2 Þk3 k1 ðk2 þ k3 Þ

ð3Þ

Taking Ks = (k− 1 + k2)/k1, Km can be re-written as: Km ¼

K s k3 Ks  ¼ ðk2 þ k3 Þ 1 þ k2

ð4Þ

k3

rearranging Eq. (4) gives:   k2 KS =Km ¼ 1 þ k3

ð4bis Þ

17

In the extreme cases (cf. Eqs. (2bis) and (4bis)), where k2< Km). The parameter b reflects the efficiency by which SpES forms products. Substrate activation occurs when b > 1, and when b < 1 there is substrate inhibition; the enzyme follows the Michaelis–Menten kinetics if b = 1 (Eq. (1)). It has been shown that the reactivity of wild-type AChE is similar with oxo-esters and homologous thio-esters, e.g. acetylcholine (ACh) versus acetylthiocholine (ASCh) [19,21,34,35]. On the other hand, wild-type BuChE was found to hydrolyze acetylthiocholine faster than acetylcholine, but it was not clear whether this was true at intermediate substrate concentration (kcat) or at high substrate concentration (b.kcat) (cf. Eq. (6)) [34,36,37]. In addition, several studies have

the apparent bimolecular constant is, kcat =Km ¼

k1 k2 ¼ k2 =KS ðk1 þ k2 Þ

ð5Þ

and if k2 < k− 1 k2 < 1Z kcat =Km < k1 ðk1 þ k2 Þ

ð5bis Þ Scheme 2.

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P. Masson et al. / Biochimica et Biophysica Acta 1774 (2007) 16–34

shown that the behavioral difference between AChE and BuChE with the ACh/ASCh couple cannot be generalized to other oxo-ester/thio-ester couples [22,36,37]. No general explanation for the relative activity of cholinesterases towards oxo-esters and thio-esters has been provided so far. Because the three-dimensional structure of human BuChE was recently solved, the loci interacting with substrate along the hydrolytic pathway are known [2]. Using the new insights into the catalytic mechanism of BuChE provided by this structural information, we re-examined the oxo-ester vs. thio-ester issue. The catalytic properties of wild type and selected mutants of human BuChE toward substrate couples were compared. A pair of positively charged substrates (BzCh vs. BzSCh) and a pair of neutral substrates (PhA vs. PhSA) were used. Mutations were either in the PAS or in the active center. In particular, we focused on mutations of residue E197 and residues in the oxyanion hole because these two sub-sites have been found to play key roles in stabilization of transition states and tetrahedral intermediates of cholinesterases [38–41]. In addition, using a mathematical analysis, we compared our data and available literature data for BuChE-catalyzed hydrolysis of various oxoester and thio-ester couples. This approach allowed us to infer the k2/k3 ratio in a limited number of cases. 2. Materials and methods 2.1. Chemicals Benzoylcholine chloride (BzCh), phenylacetate (PhA) and phenylthioacetate (PhSA) were purchased from Sigma France (Saint Quentin Fallavier, F38299). Benzoylthiocholine iodide (BzSCh) was from NCI (Tokyo, Japan). Diisopropylfluorophosphate (DFP) was from Acros Organics France (93166 Noisy-le-Grand, France). Chlorpyrifos-oxon (CPO) was obtained as standard grade from Cluzeau (33220 Ste Foy-la-Grande, France). Other chemicals were of biochemical grade.

2.2. Enzyme sources Wild-type human BuChE was from plasma or was expressed in CHO-K1 cells (ATCC, No CCL 61). Recombinant wild-type BuChE and mutants were made and expressed in transient and/or stably transfected CHO-K1 cells as previously described [17,24]. Single mutations were made at positions G117 a key residue in the oxyanion hole (G117H, G117D, G117S, G117C, G117K, G117Y), and on D70 the main residue in the PAS (D70G). Double mutations combined the mutation G117H with mutations of residues located in the vicinity of the active site serine. Three different active site positions were selected for the second mutation: a) another residue in the oxyanion hole (G115H or A199E) or near this locus (Q119E), b) the π-cation binding site (W82A, W82F), and c) residue E197 which is involved in stabilization of tetrahedral intermediates (E197D, E197Q, E197G). Natural and recombinant wild-type BuChE and mutant enzymes D70G and G117H were purified by chromatography on anion exchange gel and affinity chromatography in 20 mM potassium phosphate pH 7.0 containing 1 mM EDTA as described [17,24]. Enzyme preparations were stored at 4 °C or at − 30 °C (in the presence of 30% glycerol in buffer). The remaining mutants were either partially purified or used as non-purified, cell culture preparations. Preparations were dialyzed at 4 °C, before kinetic measurements, against appropriate buffers on Macrosep PM 10 units (Filtron Technology, Northborough, MA, USA). Buffers were either 100 mM sodium phosphate pH 7.0 or 100 mM Tris/HCl pH 7.4. Activity of enzyme preparations was assayed, according to the method of Ellman, with the standard substrate butyrylthiocholine (BuSCh) iodide at 1 mM concentration (1 unit hydrolyzes 1 μmol BuSCh in 100 mM sodium phosphate pH 7.0 at 25 °C) [42].

2.3. Kinetics of substrate hydrolysis Hydrolysis of benzoylcholine—BzCh hydrolysis by wild-type BuChE and its mutants was assayed at 25 °C in 100 mM sodium phosphate pH 7.0. The BzCh concentration ranged from 1 to 150 μM. The hydrolysis kinetics were followed by recording the decrease in absorbance at 240 nm (the difference in the extinction coefficient between substrate and products, Δε, is 6700 M− 1 cm− 1 in phosphate buffer [7,22]). Hydrolysis of benzoylthiocholine—BzSCh hydrolysis by wild-type BuChE and its mutants was assayed at 25 °C in 100 mM sodium phosphate pH 7.0. Hydrolysis of BzSCh was followed according to the method of Ellman et al. [22,42] with 0.33 mM dithio-bisnitro-benzoate (DTNB) as the chromogenic reagent by recording the increase in absorbance at 412 nm of 5-thio-2nitrobenzoate (ε = 13 300 M− 1 cm− 1) due to the reduction of DTNB by thiocholine, the hydrolysis product P1. The concentration range in BzSCh was 1 μM to 10 mM. Hydrolysis of phenylacetate—PhA hydrolysis by wild-type BuChE and its mutants was assayed at 25 °C in 100 mM Tris/HCl pH 7.4 [43]. Some experiments were carried out in buffer containing 50 mM calcium chloride as enzyme activator. Stock solutions of 0.1 and 1 M PhA were prepared in anhydrous methanol. The PhA concentration range in assays was from 0.2 to 10 mM, and the final methanol concentration was 5.3%. Hydrolysis of PhA was followed at 270 nm (ε = 1510 M− 1 cm− 1). Hydrolysis of phenylthioacetate—PhSA hydrolysis by wild-type BuChE and its mutants was assayed at 25 °C in 100 mM Tris/HCl pH 7.4, containing 0.33 mM DTNB as for the BzSCh assay. Some experiments were carried out in the presence of 50 mM calcium chloride. The temperature and buffer conditions were identical to those used for assaying the corresponding oxo-ester phenylacetate (PhA). The release of thiophenol, product P1, was monitored by recording the rate of appearance of 5-thio-2-nitrobenzoate formed in the coupled reaction of DTNB reduction. A stock solution of PhSA (150 mM) was prepared in anhydrous methanol. The PhSA concentrations in assays ranged from 30 μM to 6 mM; the final methanol concentration in cuvette was kept constant at 5% v/v. The presence of methanol in buffers has been shown to slightly affect the kinetic properties of BuChE, due to the nucleophilic competition of methanol with water. [44,45]. However, because BuChE-catalyzed hydrolysis of PhA and PhSA involves the same acyl intermediate, the presence of 5% methanol affects the deacylation rate, k3, to the same extent in both assays. The same conclusion can be drawn regarding the nucleophilicity of the buffer component Tris0. However, it should be noted that alternate nucleophiles can potentially affect the rate limiting step. Indeed, if k2 ≈ k3, the presence of methanol and/or Tris0 can switch the mechanism to k3 > k2.

2.4. Determination of catalytic parameters Because cell culture media are known to contain free thiol compounds that may interfere with the Ellman assay, non-purified enzymes were incubated in the presence of 0.5 mM DTNB for 2 h prior kinetic measurements with BzSCh or PhSA. Monitoring thiol-compound reaction with DTNB showed that consumption of free thiols occurred in less than 90 min. The steady-state catalytic parameters, Km, kcat, Kss and b were determined by non-linear computer fitting of Eqs. (1) or (6), using the Sigma Plot 4 program (Jandel Sci., San Raphael, CA, USA). In some cases, for inhibition by excess substrate when the substrate concentration range on the inhibition side was too short because of substrate solubility limit, Kss was determined according to the Haldane equation (Eq. (7)): m¼

kcat ½E 1 þ Km =½S þ ½S=Kss

ð7Þ

The active site concentration, [E], was determined by titration according to the method of residual activity using the organophosphates CPO or DFP as titrants [46]. Determination of [E] by titration with organophosphates was only possible for purified enzymes that do not hydrolyze organophosphate titrants, i.e. wild type and D70G. Active site concentration of purified G117H mutant, an OP-hydrolyzing mutant [17], was estimated from protein concentration. The protein concentration in the enzyme preparation was determined by the bicinchoninic acid method (BCA kit, Pierce, Rockford, IL, USA). One

P. Masson et al. / Biochimica et Biophysica Acta 1774 (2007) 16–34 milligram of BuChE contains 11.8 nmol of subunits. Catalytic properties of other OP-hydrolyzing mutants and certain low carboxyl-esterase activity mutants [17] were investigated on partially purified or non-purified enzymes in transient expression system medium or in stable cell culture medium dialyzed against the appropriate assay buffer. Because determination of kcat was not possible for these enzyme solutions, Vmax was expressed in units/ml. One unit hydrolyzes 1 μmol of substrate/min at 25 °C in the assay buffer (pH 7 or 7.4). For low affinity mutants, only the pseudo-first order rate constant in [S], Vmax/ Km = kcat[E]/Km, was determined from the slope of linear plots of initial velocity vs. [S].

2.5. Molecular docking of substrates in the active center of wild type and mutant BuChE Docking calculations were carried out using AutoDock version 3.0.5, with the Larmarckian genetic algorithm (LGA) [47]. The molecular models of BzCh, BzSCh, PhA and PhSA were built using the editor in WebMO [48] and minimized with MOPAC 7 (PM3 method) interfaced to WebMO. The wildtype enzyme was prepared from the crystal structure of human BuChE complexed with a molecule of choline (code pdb 1P0M). The molecules of water, sugars and all ligands were removed from the model. The catalytic serine (S198) adopts two alternate conformations in this structure; the conformation where the hydroxyl points toward the catalytic histidine (H438) was retained to perform the calculations. H438 was not protonated. G117H, G117E, G117K and G117H/W82A mutants of BuChE were built with DeepView starting from the wild type enzyme. The side chain orientation of the mutated residues was optimized with the GROMOS96 implementation of Swiss-PdbViewer [49]. The substrates and the enzyme were further prepared for the docking calculation using mglTools software from Michel F. Sanner (Scripps Research Institute). The 3D affinity grid box was designed to include the full active site gorge of human BuChE, e.g., the PAS, acyl-binding pocket, catalytic triade and cation-π interaction site. The number of grid points in the x-, y-, z-axes was 60, 60, 60 with grid points separated by 0.375 Å. Docking calculations were set to 100 runs. At the end of the calculation, AutoDock performed cluster analysis. Docking solutions with ligand all-atom root mean square deviation (RMSD) within 1.0 Å of each other were clustered together and ranked by the lowest energy representative. The lowest-energy solution was accepted as the most representative of the ES complex.

3. Results 3.1. Differences in the catalytic behavior of oxo-ester versus thio-ester The catalytic behavior of wild type BuChE and mutants towards oxo- and thio-esters showed clear differences. First, hydrolysis of BzCh by wild type BuChE and D70G was found to present a transient phase preceding the steady state [22]. The lag (τ = 3.5 min for wild-type and 1 min for D70G) was long enough to be seen under standard assay conditions and was interpreted in terms of hysteretic behavior, i.e. slow transition from an enzyme form E to E' [22,50]. Though E and E' bind BzCh, only E'S makes products. Under the same conditions, BuChE-catalyzed hydrolysis of BzSCh did not present a lag, indicating that either both ES and E'S equally make products, or that in the presence of BzSCh the conformational transition ES → E'S is rapid. These results confirm the dependence of the hysteresis on the chemical structure of substrate as previously observed. Because of the homologous structure of BzCh and BzSCh, this indicates that a subtle adjustment of the substrate in the active site at the level of acylation step is at the origin of the hysteretic behavior of BuChE. No hysteretic behavior of wild

19

type enzyme or mutants for either PhA or PhSA was observed. Second, differences in steady-state behavior were observed. The steady-state catalytic parameters of wild type and mutants for BzCh versus BzSCh are reported in Table 1, and those for PhA versus PhSA are reported in Table 2. Analysis of the data in Tables 1 and 2 indicates that the effects O → S substitution on Km and kcat (or Vmax) are due to differences in the nature of chemical bonds and interactions, and to steric factors. Let us scrutinize these data more carefully in sections 3.2a–f and 3.3. 3.2a. Hydrolysis of benzoylcholine versus benzoylthiocholine BzCh was reported to be a good positively charged substrate of wild type BuChE and D70G mutant [7,22–24]. On the other hand, the corresponding thio-ester was found to be a poor substrate for these two enzymes [22,23]. This observation is confirmed by our results for other mutants. Catalytic parameters for hydrolysis of BzCh and BzSCh by wild type BuChE and a variety of mutants are given in Table 1. BuChE and several mutants show substrate inhibition by excess BzCh and BzSCh (b < 1). For an example see Fig. 1, which shows the reaction of BzSCh with G117H b = 0.56). An exception is the reaction of G117C with BzSCh, which displays clear substrate activation (b = 2.35); see Fig. 2. This particular behavior has no clear explanation. Single mutation in the oxyanion hole at position 117 and double mutations (mutation G117H associated with mutation on another locus) caused a decrease in the catalytic activity (kcat/Km or Vmax/Km) towards both substrates, relative to wild type BuChE. This is clear for G117H, where kcat/Km is reduced by 227-fold with BzCh and 28-fold for BzSCh. Mutants for which substrate hydrolysis was first order (Table 1) support the contention also. In these cases, Km must be very large and therefore kcat/Km very small. It is also clear for all of the mutants that showed no detectable hydrolysis (indicated by n.h. in Table 1) because kcat is essentially zero and therefore kcat/Km must also be zero. Combination of high Km and low kcat cannot be ruled out either. Thus, certain substitutions of G117 and certain double mutations had deleterious effects on catalytic activity toward BzCh, BzSCh or both substrates. The oxyanion hole mutants all showed a decrease in affinity for both BzCh and BzSCh, when compared to wild type enzyme. The effect of mutations on Vmax, i.e., kcat, for hydrolysis of BzCh and BzSCh is more difficult to summarize. G117C hydrolyzes BzCh more rapidly than BzSCh, while G117H/Q119E displays an opposite pattern. G117K reacts with both substrates with the same rate. Mutants G117Y, G117H/A199E, G117H/E197G, G117H/W82F, and G117H/ W82A do not hydrolyze BzCh while they do hydrolyze BzSCh. Conversely, mutant Q119H hydrolyzes BzCh but does not hydrolyze BzSCh. For most mutants, the rate of hydrolysis can be followed up to saturation. For a few mutants, hydrolysis follows first-order kinetics in [S] (e.g. G117H/ W82A reacting with BzSCh). This indicates that their Km values are much greater than the highest BzSCh used

20

Table 1 Steady-state catalytic parameters for hydrolysis of positively charged substrates BzCh and BzSCh, by wild-type BuChE and selected mutants in 0.1 M phosphate buffer pH 7.0 at 25 °C Benzoylcholine (a)

G117S G117C G117Y G117D G117E G117K Q119H G117H/ G115H G117H/ A199E G117H/ Q119E G117H/ E197Q G117H/ E197D G117H/ E197G G117H/W82F G117H/ W82A

Benzoylthiocholine −1

−1

−1

Km μM

kcat10 min

kcat/Km 10 M

3.0 ± .3 21 ± 5 157 ± 18

14.7 ± 0.4 15 ± 1 3.4 ± 0.3

4900 ± 600 710 ± 210 21.5 ± 2.0

0.5 ± 0.1 0.4 ± 0.1 2.8 ± 0.3 59.0 ± 2.5 170 ± 10

Km μM

Vmax μmol min− 1

Vmax/Km μmol.μM− 1 min− 1

Kss mM b

n.h. 12.5 ± 1.15 146 ± 3 n.h. n.h. 18 ± 2 1415 ± 40 150 ± 20 920 ± 10 11.0 ± 0.5 100 ± 15 n.h.

6

12 ± 1

80 ± 10 6.3 ± 0.8 9.5 ± 0.5

n.h. 16.7 ± 1.6

min

Kss mM b

Km μM

Km μM n.h. 23 ± 7 220 ± 14 n.h. 110 ± 10 220 ± 20 n.h. n.h.

3

6

−1

kcat 10 min

kcat/Km 10 M

0.8 ± 0.1 1.30 ± 0.05 1.7 ± 0.2

280 ± 55 22.0 ± 1.7 9.95 ± 1.55

Vmax μmol min−1

−1

min

Vmax/Km μmol. μM− 1 min− 1

Kss mM

kcat(O/S)

(kcat/Km)(O/S)

7.3 ± 1.3

1.0 ± 0.2 0.35 ± 0.1 0.56 ± 0.04 0.9 ± 0.2

18.5 ± 2.5 11.5 ± 1.2 2.0 ± 0.4

17.5 ± 5.5 32.5 ± 11.5 2.2 ± 0.5

Kss mM

b

kcat(O/S) (c) (kcat/Km)(O/S)

3.7 ± 0.5

28 ± 3 31.75 ± 3.10− 4

1.2 ± 0.5 0.14 ± 0.01

0.9 ± 2.10− 3

1150 ± 45 1000 ± 60

10.7 ± 1.3 4.50 ± 0.70

7.1 ± 3.8 3.3 ± 2.6

first order (b) 80 ± 2

Ratio (oxoester/thioester) −1

b

Km(O/S)

0.3 ± 0.1

2.3 ± 0.2

Km(O/S)

0.55 ± 0.25

0.65 ± 0.15 0.16 ± 0.03 0.73 ± 0.03 0.70 ± 0.15

5.2 ± 0.9

9.8 ± 3.9

1.25 ± 0.08 7.40 ± 2 0.90 ± 0.05 1.4 ± 0.4

0.39 ± 0.02 0.03 ± 5.10− 3 0.40 ± 0.01

4.8 ± 0.6

500 ± 20

204 ± 3

0.40 ± 0.03

first order (b)

3.9 ± 0.1

760 ± 20

3700 ± 500

4.8 ± 0.8

0.80 ± 0.15

first order (b)

9.6 ± 0.2

530 ± 20

1700 ± 45

3.1 ± 0.3

3.1 ± 0.3

n.h.

1700 ± 70

30.5 ± 0.5

0.018 ± 0.001

n.h. n.h.

1480 ± 70 197 ± 3 first order (b)

12 ± 2

0.135 ± 0.008 4.8 ± 0.7

(a) Kinetic parameters for reaction of BzCh with wild-type BuChE and D70G are taken from ref [7,22–24]. (b) Plots of v vs. [S] were linear indicating first-order kinetics in [S], i.e., Km > [S]max where [S]max is the highest concentration used in assay. The value presented in the Vmax/Km category is the slope of the line, i.e. v = (Vmax/Km)[S] = (kcat[E]/Km)[S]. (c) kcat(O/S) is obtained by dividing Vmax for BzCh (oxo-ester) by Vmax for BzSCh (thio-ester). Vmax = kcat[E]. The amount of enzyme was the same in both sets of reactions, so [E] was the same (even though the exact concentration of enzyme was unknown). Therefore, the ratio of Vmax(o) /Vmax(s) yields kcat(o)/kcat(s), which is given as kcat(O/S). Values for (kcat/Km)(O/S) were obtained in the same fashion. n.h.: indicates no detectable hydrolysis of substrate or rates of hydrolysis less than 0.00001 ΔOD/min under experimental conditions.

P. Masson et al. / Biochimica et Biophysica Acta 1774 (2007) 16–34

Wild-type D70G G117H

3

Table 2 Steady-state catalytic parameters for hydrolysis of neutral substrates PhA and PhSA, by wild-type BuChE and selected mutants in 0.1 M Tris/HCl buffer pH 7.4 at 25 °C Phenylacetate

Wild-type D70G

2.9 ± 0.2 6.5 ± 0.6 Km mM

G117H G117S G117C G117Y G117D G117E G117K Q119H G117H/G115H G117H/A199E−Ca++ G117H/A199E+Ca++ G117H/Q119E G117H/E197Q G117H/E197D G117H/E197G G117H/W82F G117H/W82A

Phenylthioacetate

kcat 103 min− 1 32 ± 5 30 ± 2

kcat/Km 106M− 1 min− 1 11.0 ± 2.4 4.6 ± 0.7

Km mM 2.4 ± 0.1 2.8 ± 0.17

Vmax μmol min− 1 Vmax/Km μmol mM− 1 min− 1 Km mM

first order (a) 1.46 ± 0.08 1170 ± 18 6.0 ± 0.6 3280 ± 130 first order ND first order first order n.h. first order 0.70 ± 0.03 340 ± 5 0.60 ± 0.02 550 ± 5 5.8 ± 0.7 1855 ± 265 first order first order first order n.h. first order

705 ± 20 800 ± 60 545 ± 55 21.5 ± 3.0 960 ± 10 388 ± 12

first order 0.7 ± 0.1 1.6 ± 0.4 n.h. 0.23 ± 0.02

Ratio (oxoester/thioester)

kcat 103 min− 1 64 ± 10 67 ± 8

kcat/Km 106M− 1 min− 1

Kss mM

27 ± 5 24 ± 4

176 ± 17

45 ± 2 470 ± 25 900 ± 45 320 ± 35 800 ± 15 1460 ± 35 30 ± 2

first order first order first order 0.210 ± 5.10− 3 0.85 ± 0.15 3.6 ± 0.2 first order first order first order

115.0 ± 1.5 390 ± 70 2060 ± 70

1010 ± 55

4.45 ± 0.10

17130 ± 300

2130 ± 45 1470 ± 310 920 ± 240 750 ± 70 3300 ± 20 1540 ± 20 55 ± 1 90 ± 2 545 ± 20 460 ± 10 575 ± 55 3000 ± 30 3130 ± 30 81 ± 1 345 ± 15 3860 ± 175

Km(O/S)

kcat

(O/S)

1.2 ± 0.1 0.5 ± 0.1 2.32 ± 0.35 0.45 ± 0.08

Vmax μmol min− 1 Vmax/Km μmol mM− 1 min− 1 Kss mM 970 ± 60 735 ± 195

b

b

5.1 ± 0.6

2.6 ± 0.8

3.8 ± 0.8

8.6 ± 2.5

Km(O/S) 2.18 ± 0.43 3.75 ± 0.95

kcat(O/S) 1.2 ± 0.1 4.5 ± 0.1

(kcat/Km) (O/S) 0.4 ± 0.1 0.20 ± 0.06 (kcat/Km)(O/S) 0.33 ± 0.01 0.55 ± 0.15 0.6 ± 0.2

0.290 ± 0.005 0.25 ± 0.01

0.70 ± 0.02 1.90 ± 0.08

3.4 ± 0.2 2.95 ± 0.08 0.75 ± 0.15 1.45 ± 0.30 1.6 ± 0.3 0.90 ± 0.15

0.50 ± 0.03 0.86 ± 0.08 1.95 ± 0.45 0.55 ± 0.10 0.27 ± 0.01 0.47 ± 0.01 0.40 ± 0.03 0.260 ± 0.025

P. Masson et al. / Biochimica et Biophysica Acta 1774 (2007) 16–34

Km mM

(a) First-order kinetics in [S], cf. footnote in Table 1. n.h., indicates no detectable hydrolysis of substrate or rates of hydrolysis less than 0.00001 ΔOD/min under experimental conditions. ND, not determined.

21

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P. Masson et al. / Biochimica et Biophysica Acta 1774 (2007) 16–34

(10 mM), and that saturation was not reached under experimental conditions. Comparison of the specificity constants, kcat/Km or Vmax/Km is more consistent. The specificity constant for BzCh is generally larger than that for BzSCh, for both wild type and mutants, indicating that the oxo-ester is much better hydrolyzed than the thio-ester. There is an exception in the case of the double mutant G117H/E197Q. The (kcat/Km)(O/S) ratio, R, is related to the difference in ‡ transition state free energies (ΔΔGES = − RTLnR) for acylation of oxo- versus thio-ester (cf. Eq. (5)). From the values of R, it can be concluded that the efficiency by which wild type and mutant are acylated is better for BzCh than for BzSCh. For wild type, there is a difference in transition state energy of 1.7 kcal mol− 1. However, mutations in the oxyanion hole tend to ‡ decrease the relative ratio R. ΔΔGES drops to 0.5 kcal mol− 1 for G117H. Thus, mutations in the oxyanion hole have a less deleterious effect on the efficiency by which mutants are acylated by BzSCh than acylated by BzCh. 3.2b. A general strategy for calculation of the ratio of acylation (k2) and deacylation (k3) rate constants for BuChE In this section, we will describe a general scheme for determining the relative rates of acylation and deacylation of BuChE from the experimentally accessible kinetic parameters Vmax, Km and Ks. The concept exploits the relationships between these parameters for two homologous substrates and is similar to the protocol described by Brestkin and coworkers [51]. It is critical that a pair of substrates be used which generate the same acyl intermediate on the active center serine. We will use BzCh and BzSCh as examples for the derivation. The common acyl intermediate for these substrates is benzoyl-serine. After deriving the equations for the scheme, its utility will be illustrated on wild type BuChE. Then we will extend the concept to mutant forms of BuChE. Finally, we will illustrate the application of the scheme to other oxo-ester/thioester pairs.

Fig. 1. A plot of rate ΔA412/min versus BzSCh concentration for the hydrolysis of BzSCh by the G117H mutant of BuChE in 0.1 M phosphate buffer, pH 7.0 at 25 °C. See Materials and methods for experimental details.

Fig. 2. A plot of rate ΔA412/min versus BzSCh concentration for the hydrolysis of BzSCh by the G117C mutant of BuChE in 0.1 M Tris buffer, pH 7.4 at 25 °C. The enzyme was pre-incubated with DTNB to neutralize possible thiol compounds present in the medium, see Materials and methods for experimental details.

The derivation starts from the ratio of (Vmax/Km)O/(Vmax/ Km)S, the left-hand expression in Eq. (8), where the subscript S stands for the thio-form and the subscript O stands for the oxoform. Substituting Vmax = kcat [E] from Eq. (2) gives the middle expression in Eq. (8). If same amount of enzyme preparation is used for the determination of Vmax and Km with both substrates, the [E] terms will cancel. Then, k2/Ks can be substituted for kcat/ Km (from Eq. (5)) to yield the right-hand term in Eq. (8): ðVmax =Km Þoxo ðkcat ½E=Km Þoxo Ks;s k2:o ¼ ¼ ðVmax =Km Þthio ðkcat ½E=Km Þthio Þ k2;s Ks;o

ð8Þ

where Ks,o and Ks,s are the dissociation constants of ES in which S is oxo- and thio-ester, respectively, and k2,o and k2,s are the acylation rate constants for oxo- and thio-ester, respectively. It should be noted that Eq. (8) is valid if kcat/Km is not partially rate limited by diffusion. If diffusion is ratelimiting, then, kcat/Km ≈ k1. Such a situation, that implies k−sub 1 < k2, would occur only for very fast substrates with kcat/ Km > 109 M− 1 s− 1 at very low ionic strength [19]. Thus, the argument that kcat/Km is not limited by diffusion probably does not apply to BzCh where kcat/Km = 8.107 M− 1 s− 1 (cf. Table 1). The objective of this portion of the derivation is to obtain a value for the k2,o/k2,s ratio in terms of measurable quantities. In order to proceed, it is necessary to determine the dissociation constants for both substrates, Ks,o and Ks,s. Allow the ratio Ks, o/Ks,s to be X. Substituting X for Ks,o/Ks,s in Eq. (8) and rearranging gives Eq. (9), from which the term β = k2,o/k2,s is defined. The terms on the right hand side of Eq. (9) are all experimentally determined; therefore, β is experimentally determined.

b¼

ðkcat =Km Þoxo k2:o ¼ Xd k2;s ðkcat =Km Þthio Þ

ð9Þ

P. Masson et al. / Biochimica et Biophysica Acta 1774 (2007) 16–34

23

Fig. 3. Dependence of k2/k3 on the value of β for thio- and oxo-esters. The lines are theoretical plots for various values of a. The horizontal line at k2/k3 = 1 in each panel, indicates the demarcation between acylation and deacylation as rate-limiting step. Panel A: thio-ester with a < 1 and β < a; (k2,s/k3) = [1/(1 − a)][(a/β) − 1]. Line a = 0.1 illustrates the possibilities available for substrate (L(+)AβMSCh). Line a = 0.5 illustrates the possibilities available for substrate (PhSA). In both cases, determination of the nature of k2/k3 is inconclusive. Panel B: thio-ester with a > 1 and β > a; (k2,s/k3) = [1/(1 − a)][(a/β) − 1]. Lines a = 1.1, a = 1.2, a = 1.3 illustrate the possibilities available for substrates (BuSCh), (ASCh), and (PrSCh), respectively. For these cases, determinations of the nature of k2/k3 are inconclusive. Line a = 2.5 illustrates the result for a value of a > 2, where acylation is always rate limiting. Panel C: oxo-ester with a < 1 and β < a; (k2,o/k3) = [1/(1 − a)][a − β]. Line a = 0.1 illustrates the possibilities available for substrate (L(+)AβMCh), where acylation is rate limiting. Line a = 0.5 illustrates the possibilities available for substrate (PhA), where acylation is rate limiting. Line a = 0.8 illustrates a general case for which determination of the nature of k2/k3 are inconclusive. Panel D: oxo-ester with a > 1 and β > a; (k2,o/k3) = [1/(1 − a)][a − β]. Lines a = 1.1, a = 1.2, a = 1.3, and a = 1.7 illustrate the possibilities available for substrates (BuCh), (ACh), (PrCh) and (ScdCh), respectively. For these cases, determinations of the nature of k2/k3 are inconclusive.

Eq. (10) is the ratio of the kcat expressions for the oxo- and thio-esters (Eq. (2)). a¼

kcat:o k2;o ðk2;s þ k3 Þ ¼ kcat;s k2;s ðk2;o þ k3 Þ

ð10Þ

Since the substrate pair was chosen such that the acylintermediate would be the same for each substrate, k3 is the same for both the oxo- and thio-expressions. Since the same amount of enzyme was used in the determination of the kinetic constants, Vmax (which equals kcat[E]) could be used in place of kcat for each substrate. If we allow kcat,o/kcat,s = a, and k2,o/ k2,s = β, re-arrangement of Eq. (10) leads to the following expressions for k2,s/k3 and k2,o/k3: k2:s ½ða=bÞ  1 ¼ 1a k3

ð11Þ

k2:0 ða  bÞ ¼ 1a k3

ð12Þ

Eqs. (11) and (12) are valid only when the mathematical expressions for the k2/k3 ratios are non-negative. Eq. (11), k2,s/k3 vs. β, is a rectangular hyperbolic function with two asymptotes, k2,s/k3 → ∞ as β approaches 0 and k2,s/k3 → − 1/(1 − a) as β approaches infinity. The latter k2,s/k3 limit is negative if a < 1 (Fig. 3A) and positive if a > 1 (Fig. 3B). Eq. (12), k2,o/k3 vs. β, is a straight line with slope = − 1/(1 − a) and intercepts at k2,o/k3 = a/(1 − a) and β = a (refer to Fig. 3C for a < 1 and Fig. 3D for a > 1). If k2/k3 in Eqs. (11) and (12) is set equal to 1, then the equations can be solved for β in terms of a (Table 3). By definition, when k2/k3 > 1, deacylation is rate-limiting; when 0 < k2/k3 < 1, acylation is rate-limiting. This occurs only when a < 1 and β < a, or when a > 1 and β > a, because those are the conditions under which Eqs. (11) and (12) are non-negative. Under these circumstances, the ratios of k2/k3 can be evaluated by applying the rules outlined in Table 3. In general, as the value of a approaches the value of β the numerators in Eqs. (11) and (12) become smaller than the denominators, the k2/k3 ratios become less than one, and acylation becomes more rate limiting. In the particular case

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P. Masson et al. / Biochimica et Biophysica Acta 1774 (2007) 16–34

Table 3 Determination of the rate-limiting step for substrate hydrolysis from the study of cholinesterase-catalyzed hydrolysis of oxo-versus thio-ester pairs a < 1 and β < a

a > 1 and β > a

k2,s/k3 =

(a/β − 1)/(1 − a)

(1 − a/β) / (a − 1)

rate-limiting step with thio-ester is

if β < a/(2 − a) deacylation

k2,o/k3

(a − β) / (1 − a)

rate-limiting step with oxo-ester is

if a ≤ 0.5, any β acylation

if β > a/(2 − a) acylation

if a > 2, any βacylation

if 1 < a < 2 if β < a/(2 − a): acylation if β > a/(2 − a): deacylation

(β − a) / (a − 1) if a > 0.5 if β > 2a − 1: acylation if β < 2a − 1: deacylation

if β < 2a − 1 acylation

if β > 2a − 1 deacylation

a = kcat, O/kcat, S. β = k2,o/k2, S.

where a = β, acylation for both substrates is absolutely rate limiting. On the other hand, as the value of a approaches 1, the denominators in Eqs. (11) and (12) become smaller than the numerators, the k2/k3 ratios become larger than one, and deacylation becomes more rate limiting. If a = 1, then deacylation for both substrates is absolutely rate limiting. More specifically, for the oxo-esters (Eq. (12)), when β = 2a − 1, i.e. k2,o = k3, then acylation equals deacylation, i.e. k2,o = k3, and both are partly rate-limiting (horizontal line for k2,o/k3 = 1 in Fig. 3C, D). If a > 1, when β < 2a − 1, then acylation is more rate-limiting, i.e. k2,o/k3 < 1, and when β > 2a − 1, then deacylation is more rate-limiting, i.e. k2,o/k3 > 1. If a < 1, the situation is just the opposite: when β < 2a − 1, then deacylation is more rate-limiting, i.e. k2,o/k3>1, and when β > 2a − 1, then acylation is more rate-limiting, i.e. k2,o/k3 < 1. For the thio-esters (Eq. (11)), when β = a/(2 − a), i.e. k2,s = k3, acylation equals deacylation and both are partly rate-limiting, i.e. k2,s = k3 (horizontal line for k2,s/k3 = 1 in Fig. 3A, B). If a < 1, when β < a/(2 − a), deacylation is more rate-limiting, i.e. k2,s/k3>1, and when β > a/(2 − a), acylation is more rate-limiting, i.e. k2,s/k3 < 1. If a > 1, acylation is more rate-limiting when a > 2, and deacylation is more rate-limiting when 1 < a < 2. All cases are summarized in Table 3. Given Vmax/[E]o for both the oxo- and thio-substrate, the value for a can be calculated. Given Ks and Vmax/[E]oKm for both the oxo- and thio-substrate, the value of β can be calculated. With both a and β, the k2/k3 ratio for both substrates can be determined using the rules above. In the event that the kcat values are known, direct calculation of both the acylation and deacylation rate constants, k2 and k3, is possible, using Eq. (2bis) and the experimentally determined ratios for k2/k3 from Eqs. (11) and (12). Thus, it is possible to determine the ratio of the acylation rate to the deacylation rate for BuChE by measuring Vmax, Km and Ks with a pair of homologous substrates, for which the acyl-intermediate is the same. In practical terms, the limitation of the method is the need for a value for Ks. If substrate binding is rapid equilibrium, then k2 < k− 1 (cf. Scheme 1) and therefore, Ks = k− 1/k1. This situation occurs with slow substrates such as PhA and PhSA (kcat/Km ≈ 105 M− 1 s− 1; Table 2). Under these conditions, the Ki for an isosteric inhibitor may be determined and used as a reasonable surrogate for Ks. For a fast substrate like BzCh (kcat/Km ≈ 108 M− 1 s− 1; Table 1), Ks must be rigorously taken as (k− 1 + k2)/k1. This makes Ks = Ki(1 + k2/k− 1) and a value for k2/k− 1

must be obtained to use with the surrogate Ki. The ratio k2/k− 1 can be estimated using different methods. Solvent perturbation methods have been used to determine k2/k− 1 in cholinesterase-catalyzed reactions. The dependence of kcat/Km on viscosity, for reactions carried out in the presence of increasing concentrations of viscosogens leads to estimates of k2/k− 1 [52]. However, the osmotic effect of viscosogens may alter the solvation of the enzyme active center [41] by sucking out water molecules from the gorge. This may change the kinetic behavior, altering the enzyme conformation and/or of its ligand binding capacity and chemical activity. For example, we found that the D70G mutant of BuChE displays activation by excess substrate in the presence of polyols while it does not in simple buffer [28]. Similar effects would be expected for other perturbants, such as organic solvents or heavy water. In theory, other methods that do not require solvent perturbation by viscosogens, a co-solvent [44,45] or heavy water [19] can be used to determine individual rate constants. For example, the temperature dependence of the Michaelis–Menten parameters can provide values of k1, k− 1, k2 and k3 when there is nonlinearity in Arrhenius plots [53]. Analysis of the decay of progress curves can theoretically lead to determination of k1, k− 1 and k2 if [E] is known [54]. However, these methods are not easy to apply and experimental errors introduced in such analyses lead to erroneous values for the rate constants. Thus, Ks is difficult to determine with accuracy. Then, for most oxo-ester/thio-ester pairs, Ks values are not known (cf literature data from Table 4). Therefore, determination of β from Eq. (9) is not possible. However, if Ks is not known, a study of the variation of k2,o/k3 and k2,s/k3 as a function of β, allows for the determination of particular values of β where the rate-limiting step changes for both substrates. Then applying mathematical inequality conditions to Eqs. (11) and (12), the limit values of k2,o/k3 and k2,s/k3 can be determined in some cases from the relationship between β and the k2/k3 ratios as follows (refer to Table 3 and Fig. 3). (a) Thio-esters with a < 1 and β < a. Fig. 3A shows the dependence of k2,s/k3 as a function of β for different values of a < 1. Thus, for thioesters, when a < 1, determination of whether acylation or deacylation is rate limiting is possible only when values for both a and β are available. (b) Thio-esters with a > 1 and β > a. Fig. 3B shows the dependence of k2,s/k3 as a function of β for different

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25

Table 4 Catalytic constants and acylation/deacylation rate constants of wild-type BuChE for pairs of oxoester/thioester giving the same acyl intermediate EA, pH = 7.0, 25 °C P1 PhA PhSA ACh ASCh PrCh PrSCh BuCh BuSCh BzCh BzSCh L(+)AβMCh L(+)AβMSCh SdCh Ch SdSCh thioCh (−)cocaine (−)thiococaine (+)cocaine

phenol thiophenol choline thiocholine choline thiocholine choline thiocholine choline thiocholine Aβcholine Aβthiocholine choline

EA

acetyl acetyl acetyl acetyl propionyl propionyl butyryl butyryl benzoyl benzoyl acetyl acetyl succinyl 600 [7] thiocholine succinyl 350 [24] (−)ecgonine methyl ester benzoyl (−)thioecgonine methyl ester benzoyl (+)ecgonine methyl ester benzoyl 7 500 [64] – (+)thiococaine (+)thioecgonine methyl ester benzoyl

kuncat (min− 1) −5

kcat (min− 1)

0.13 × 10 [66]

5.52 × 10− 5

1.7

32,000 64,000 13,000 (a) [7] 11,000(a‘) [62] 25,000 (a) [7] 19,500(a‘) [62] 26,000 (a) [7] 24,000(a‘) [62] [67] 14,700 [22] 800 [22] 1500 (b) [68] 14,000(c) [34] >1.7

kcatO/kcat,S = a k2,o/k2,s = β 0.5 0.5 1.2 1.2 (d) 1.3 1.3 (d‘) 1.1 1.1(d“) 18.4 18.4 0.1 0.1

(f)

limk2/k3 (g) k2 (min− 1) k3 (min− 1)

<0.5 <0.5 >1.2 >1.2 >1.3 >1.3 >1.1 >1.1 74* 74* <0.1 <0.1

<1 U U <5, U U <3.3, U U <10, U 3 0.04 <0.11 U

20* 20*

0.0002 0.00001

58,800 830

19,800 20,800

3.9 0.24

19,800(e) 19,800(e)

ND

19,800

U

1.7 >1.7 8.93 × 10− 5 [65] 3.9 [63] 0.24 [64] 8.93 × 10− 5 [65] – 0.6 ND

<1.4, U 16.2 16.2 12 100 ND

19800(e)

(a) turnover numbers were recalculated taking into account that numbers presented in [7] were bkcat instead of kcat (cf. Eq. (9)). (a') calculated from [24,62]. (b) 5% of Vmax with BuCh [68]. (c) 90% of Vmax with BzCh or 60% of Vmax with ASCh [22]. (d–d’’) ratios calculated from data in ref. 37 are 0.8 ; 1.0 and 1.3, respectively. (e) k3 values are from for enzyme debenzoylation [22]. (f) limiting values of β were calculated from Eqs. 14–15. (g) lim k2/k3 indicates the upper limit of this ratio. * indicates experimentally determined β values. U, “undecidable” rate limiting step.

values of a > 1. If 1 < a < 2, it is impossible to decide whether k2,s/k3 is greater or less than 1 without the additional determination of β. However, if a > 2, then k2,s/ k3 is always less than one, and acylation is always rate limiting. Thus, for the thio-ester reaction acylation is always rate limiting when kcat,O/kcat,S > 2. (c) Oxo-esters with a < 1 and β < a. Fig. 3C shows the dependence of k2,o/k3 as a function of β for different values of a < 1. Thus, in general, determination of whether acylation or deacylation is rate limiting is not possible on the basis of a alone, rather it requires values for both a and β. However, if the experimental value of a is lessthan-or-equal-to 0.5, then k2,o/k3 will be always less than one and acylation is always rate-limiting. (d) Oxo-esters with a > 1 and β > a. Fig. 3D shows the dependence of k2,o/k3 as a function of β for different values of a > 1. If β < (2a − 1) then k2,o/k3 < 1, and acylation is rate limiting. If β > (2a − 1) then k2,o/k3 > 1, and deacylation is rate limiting. Thus, again determination of whether acylation or deacylation is rate limiting is not possible on the basis of a alone, rather it is possible only when values for both a and β are available.

easy to determine a because it is the ratio of kcat,O/kcat,S (or Vmax,o/Vmax,s, if the same concentration of enzyme is used in all assays). However determination of β is more difficult, generally requiring measurement of the Ks values for both the oxo- and thio-ester. There are only two conditions that allow unencumbered prediction of the ratio of k2/k3 on the basis of a alone: (1) For the thio-ester, when kcat,O/kcat,S>2, then the k2,s/k3 ratio will always be less than one (Fig. 3B) and acylation will always be rate limiting. (2) For the oxo-ester, when kcat,O/kcat,S ≤ 0.5, then the k2,o/k3 ratio will always be less than one (Fig. 3C) and acylation will always be rate limiting. In addition, if experimental error is taken into consideration, i.e. if the difference a-β is less than the error of a, some values of a may allow an estimation of the k2/k3 ratio in general cases. This strategy was applied to (a) BzCh/BzSCh and PhA/ PhSA with wild type BuChE and mutants; and (b) other pairs of oxo-/thio-esters, starting from literature data for their Vmax or kcat values (Table 4).

In summary, under most conditions, determination of whether acylation or deacylation is rate limiting is possible only when values for both a and β are available. It is relatively

To illustrate this analysis, the reaction of wild-type BuChE with BzCh and BzSCh will be developed. Values for kcat and Km with BzCh are 1.47 × 104 min− 1 and 3.0 μM, respectively;

3.2c. Application of the scheme for determination of the ratio for k2 and k3 to wild type BuChE reacting with BzCh and BzSCh as the oxo-ester/thio-ester couple

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with BzSCh they are 8 × 102 min− 1 and 2.8 μM (Table 1). Ks values of wild-type BuChE for BzCh and BzSCh were estimated as 14.1 μM (Ks,o) and 3.3 μM (Ks,s), respectively [22]. The ratio Ks,o/Ks,s equals 4.27. Substituting these measured kinetic parameters into Eqs. (9) and (10) yield values for a = 18 and β = 74. From Eq. (12), k2,o/k3 equals (18–74)/(1– 18) = 3, and from Eq. (11), k2,s/k3 equals ((18/74) − 1))/(1–18) = 0.04. Finding that k2,o/k3 = 3 indicates that both k2,o and k3 are partially rate limiting for the hydrolysis of BzCh, with deacylation (k3) being the more rate-limiting of the two. Finding that k2,s/k3 = 0.04 indicates that k2,s is the primary rate limiting step for the hydrolysis of BzSCh. These results are consistent with a previous study, where it was found that the rate-limiting step for hydrolysis of BzCh by wild type BuChE was deacylation (k3 < k2) while the rate-limiting step was acylation for BzSCh (k2 < k3) [22]. kcat values for BzCh and BzSCh with wild type BuChE are 14 700 min− 1 and 800 min− 1, respectively (Table 1). Values for k2 and k3 can be calculated from Eq. (2bis) and the k2o/k3 = 3.0 and k2s/k3 = 0.04 ratios obtained from Eqs. (11) and (12). These calculations yield a value for k2 with BzCh of 58 800 min− 1 and for BzSCh of 830 min− 1. A value for k3 of 19 800 min− 1 is obtained for BzCh and a value of 20,800 min− 1 is obtained for BzSCh. These values are comparable to those reported in reference [22]. If Ks were not known, determination of β from Eq. (9) would have not been possible. However, by applying the rules from Table 3, the limiting values of the k2/k3 ratios for both substrates can be evaluated, as follows. Table 3 and Fig. 3B show that for the thioester, when a > 2, acylation is always rate limiting. The value of “a’ is 18, therefore acylation is rate limiting for BzSCh. Fig. 3B shows that as a and β both increase, the k2,s/k3 ratio decreases. At a = 2.5 and β = 9 the k2,s/k3 ratio is 0.5. Qualitative extrapolation to a = 18 and β > 18 suggests that k2,s/k3 will be substantially lower than 0.5. This value is in qualitative agreement with the experimentally determined value of 0.04. Evaluation of the oxo-ester, BzCh, is less definitive. Since a > 1, then β > a. However, if β < 35 acylation would be rate limiting, whereas if β > 35 deacylation would be rate limiting. With no way to estimate the actual value of β, it is not possible to determine whether acylation or deacylation will be rate limiting. Only the knowledge of Ks, leading to an experimental value β = 74, allows to estimate k2/k3 = 3, i.e. acylation as the rate limiting step ([22] and Table 4). 3.2d. Extension of the scheme for determination of the k2/k3 ratio to the mutants G117H and G117C with BzCh/BzSCh With G117H, it was possible to measure kcat and Km for BzCh and BzSCh, but not Ks. As a consequence, the Ks,o/Ks,s ratio was estimated from the values for Km, using the following rationale. Km values of the G117H mutant for BzCh and BzSCh are 157 μM and 170 μM, respectively. Corresponding values for wild type BuChE are 3 μM and 2.84 μM. Both Km values for G117H show similar increases of 50- to 60-fold compared to Km values of wild type. Thus, it can be reasonably assumed that the G117H mutation in the oxyanion hole caused a parallel

decrease in binding affinity (expressed as Ks) for BzCh and BzSCh. It follows that the ratio Ks,o/Ks,s should be 4.27 for G117H, the same as for wild type. Using 4.27 in Eq. (9) together with kcat/Km values of 21.5 × 106 M− 1 min− 1 for BzCh and 9.95 × 106 M− 1 min− 1 for BzSCh (see Table 1) yields a value for β (k2,o/k2,s) for G117H of 9.2. The ratio of kcat,O/kcat,S gave a value of 2.0 for the a-term. Substitution of these a and β values into Eq. (12) indicated that hydrolysis of BzCh by the G117H mutant occurs with a k2,o/k3 ratio of 7.2, i.e., deacylation is rate-limiting (k2 > k3). For hydrolysis of BzSCh by this mutant (Eq. (11)), k2,s/k3 = 0.8 ± 0.4. This ratio is statistically not different than unity, indicating that within experimental error acylation and deacylation are equally rate limiting. In addition, these ratios are 2.5- and 20fold higher than the corresponding ratios for wild-type BuChE. This indicates that the mutation G117H in the oxyanion hole has differently altered the enzyme catalytic machinery for both catalytic steps. The fact that kcat of G117H for BzSCh is higher than that of wild-type while kcat for BzCh is lower (Table 1), demonstrates that mutation G117H caused a considerable increase in the acylation rate for the thio-ester. Since kcat was determined, values for k2 and k3 could be calculated from Eq. (2bis). For hydrolysis of BzCh by G117H, k2,0 equals 27 900 min− 1, and for hydrolysis of BzSCh by G117H, k2,s equals 3000 min− 1. The deacylation rate constant, k3, equals 3800 min− 1 for both. These results clearly show that mutation G117H reduced the acylation rate constant for hydrolysis of BzCh by 2-fold, while this rate was increased 4fold for hydrolysis of BzSCh. The value of k3 for both substrates, same acyl intermediate, was 5-fold reduced by the mutation. The Ks values for BzCh and BzSCh were not directly measured for G117H. However, these values can be calculated from the measured Km values by substituting the k2/k3 ratios from Eqs. (11) and (12), into Eq. (4). In this way, estimates of Ks for G117H were calculated to be 1300 μM with BzCh and 300 μM with BzSCh. For G117C, Km values for BzCh and BzSCh are 4- to 8fold higher than the corresponding Km values for wild type BuChE. Therefore, using the same logic that was applied to G117H, it is reasonable to assume that Ks,o/Ks,s is 4.27. If this is used for the value of X in Eq. (9), then estimates of k2,s/ k3 = 0.2 and k2,o/k3 = 9 can be made. These values are qualitatively similar to those for wild type BuChE and G117H, indicating that acylation is rate limiting for BzCh and deacylation is rate limiting for BzSCh. This suggests that mutation G117C did not change the catalytic mechanism compared to G117H and wild-type BuChE. Further calculations of the kinetic parameters for G117C are precluded by the absence of values for kcat. 3.2e. Extension of the scheme for determination of the ratio of k2 and k3 to the other oxyanion hole mutants with BzCh/BzSCh For the other oxyanion hole mutants that we tested, only Vmax, Km, and Vmax/Km data were obtained (Table 1). If for these mutants, the Ks,o/Ks,s ratio X were equal to 4.27 as for the

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G117H mutant and wild-type enzyme, then the k2,o/k2,s ratio (β) could be calculated. However, for these mutants, Km values for BzCh and BzSCh did not change in the same ratio as is seen for wild type BuChE (Table 1). Because Km is a composite constant (Eq. (4)), this suggests that taking the ratio Ks,o/Ks,s as 4.27 might be erroneous, which in turn implies that k2,o/k2,s ratios calculated using X = 4.27 are likely to be erroneous. These results highlight the limitation of the calculation method. A reliable estimate of the Ks ratio, X, is needed. Without direct measurement of Ks,o and Ks,s or a clear parallel to Km changes for wild type, the method may not be applied. 3.3. Hydrolysis of phenylacetate vs. phenylthioacetate As expected with a neutral ester, wild type BuChE and its mutants all were found to obey the Michaelis–Menten model with PhA. A similar behavior was found with PhSA. In particular the D70G mutant behaves as wild type; this is because residue D70 in the PAS is not involved in initial binding of neutral substrates. However, several mutants (G117C, G117D and G117H/A199E) displayed substrate activation with this thio-ester, i.e. b > 1 (Table 2, Fig. 4). Such a behavior cannot be explained by the mechanism that involves binding of a second substrate molecule on the PAS [25–27]. Rather, this behavior supports previous results indicating that substrate binding on the PAS is not the sole determinant of activation by excess substrate [28–31] and that activation is not dependent on the substrate charge [55]. Actually, substrate activation with PhSA was already reported with horse BuChE [56] and rabbit liver BuChE [57]. This behavior was interpreted in terms of a change in the rate-limiting step, i.e., an increase of k3 with substrate concentration such that at low substrate concentration k2 > k3, while at high concentration k2≈k3 [58]. Accordingly, the authors proposed that at high concentration, a second substrate molecule binds to the acylated enzyme and thereby accelerates deacylation.

Fig. 4. Hydrolysis of PhSA by the G117H/A199E mutant of BuChE in 0.1 M phosphate buffer, pH 7.0 at 25 °C (enzyme was pre-incubated with DTNB to neutralize possible thiol compounds present in the medium, see Materials and methods for experimental details).

27

Because wild type and D70G human enzymes are Michaelian with PhSA, the fact that other cholinesterases and the above-mentioned mutants (G117C, G117D and G117H/A199E) show substrate activation with PhSA and other neutral esters can be tentatively interpreted as the result of the binding of a second substrate molecule into the active site gorge. This second molecule may bind close to the acylated serine (S198). Interestingly, the G117H/A199E mutant had a Michaelian behavior with PhSA in the presence of 50 mM Ca+. Under these conditions, its catalytic parameters were similar to the catalytic parameters of substrate-activated enzyme (Table 2). This suggests that Ca+ may bind to the same location as the putative second substrate molecule and accelerate deacylation throughout the whole substrate concentration range. Unlike what was observed for hydrolysis of BzCh and BzSCh, catalytic parameters for hydrolysis of PhA and PhSA were similar for wild type BuChE and the D70G mutant (Km of the same order and same kcat,O/kcat,S ratio, a≈0.5). On the other hand, mutations in the oxyanion hole dramatically altered catalytic parameters for hydrolysis of both esters (Table 2). For example, affinity was so reduced that hydrolysis of both esters by a variety of mutants was first-order in [S] under our experimental conditions. Double mutation can compensate for the deleterious effect of mutation G117H for hydrolysis of both substrates (e.g., G117H/A199E and G117H/Q119E, cf. Table 2). Unlike the situation that prevails for BzCh vs. BzSCh, kcat/ Km ratios and Vmax/Km ratios show that the thio-ester (PhSA) is a better substrate than the oxo-ester (PhA) for most all of the enzymes tested: wild type, D70G and oxyanion hole mutants (Table 2). There were however two interesting exceptions: the G117Y mutant did not hydrolyze PhSA while it hydrolyzed PhA, and the Q119H mutant did not hydrolyze PhA while it hydrolyzed PhSA. The behavior of both mutants was opposite with the BzCh/BzSCh couple. Because no hypothesis can be made about the Ks,o/Ks,s ratio for PhA/PhSA, estimation of the values for k2,s/k3 and k2,o/k3 generally was not possible. However, the limiting values for these ratios can theoretically be calculated from Eqs. 11–12, and thus the rate-limiting step can be estimated (Table 3). In principle, such a calculation allows quantification of the effect of any mutation on hydrolysis of this oxo-ester, thio-ester pair in terms of k2/k3. With wild type BuChE, a = 0.5. From the rules in Table 3, where a < 1 and β < a, Eq. (11) shows that determination of the rate-limiting step for PhSA is not possible without determination of β. Combining the limiting conditions with Eq. (12) tells us that 0 < a − β < 0.5 and 1/(1 − a) = 2. Consequently, k2,o/k3 is always less than one. Thus, for hydrolysis of PhA, the ratelimiting step is acylation (Fig. 3C). This conclusion agrees with that of Volkova [59], which was obtained by Arrhenius plot analysis of BuChE-catalyzed hydrolysis of PhA. It is noteworthy that with the same substrate, AChE was found to show opposite behavior, deacylation being the rate-limiting step [60,61]. Mutations in the oxyanion hole are more deleterious for the hydrolysis of PhA, the oxo-ester, than for the hydrolysis of PhSA, the thio-ester. Because several oxyanion hole mutants

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display a > 1 (Table 2), i.e. β > a, for this pair of substrates. Therefore, the ratios, (kcat/Km)O/(kcat/Km)S = β.Ks,s/Ks,o < 1, which suggests that Ks,s < Ks,o for these mutants. This means that mutation on G117 leads to an increase in affinity for PhSA compared to PhA, e.g. for G117C. This undoubtedly reflects differences in the adjustment of enzymes and substrate to form ES in the mutated active site, due to steric constraints imposed by the O → S. Finally, as for the previous pair of substrates, BzCh vs. BzSCh, it may be concluded that mutations in the oxyanion hole and vicinity are less deleterious for hydrolysis of the thio-ester than for its homologous oxo-ester. 3.4. Effects of replacing the ethereal O by S on acylation and deacylation rates of other pairs of oxo- and thio-esters Comparison of data for BzCh/BzSCh, PhA/PhSA, and other pairs of oxo/thio esters can be made using Eqs. (11) and (12), and following the rationale that was applied for PhA/PhSA (Table 4). Calculation for acyl-choline/acyl-thiocholine esters (acyl: acetyl, propionyl, butyryl) led to inconclusive results (Table 4). However, hyperbolic plots of k2,s/k3 vs. β (Fig. 3B) show that (1) acylation might occur as the rate-limiting step for the thio-substrate in narrow intervals on β-axis (a − β) = 0.1 for BuSCh,= 0.3 for PrSCh = 0.2 for ASCh; where β1 is the solution of Eq. (11) when k2,s/k3 = 1; and (2) for large values of β, asymptotic values of k2,s/k3 are 3.3 for PrSCh, 5 for ASCh and 10 for BuSCh. Thus, inspection of hyperbolic plots suggests either that acylation and deacylation are partly ratelimiting or that deacylation is rate limiting for these thioesters. Previous determinations obtained by Arrhenius plot analysis [56] or nucleophilic competition between water and methanol for deacylation [45] showed that deacylation is the rate-limiting step for hydrolysis of acyl-thiocholinesters. For hydrolysis of acyl-cholinesters, in the present study, calculations cannot determine whether acylation or deacylation is rate-limiting. However, linear plots of k2,o/k3 vs. β (Fig. 3D) show that acylation would be the rate-limiting step in only very narrow intervals on β-axis (a − β) = 0.1 for BuCh,= 0.3 for PrCh = 0.2 for ACh; where β1 is the solution of Eq. (12) when k2,o/k3 = 1. Thus, it is likely that acylation and deacylation are partly rate-limiting or that deacylation is fully rate-limiting with β1 > 1.2 for BuSCh, > 1.6 for PrCh and > 1.4 for ACh. Previous work suggested that deacylation was the rate-limiting step (k2/k3 = 5.2) for hydrolysis of ACh by horse BuChE [51,59]. For AChE from Electrophorus electricus, Froede and Wilson determined that acylation was slightly faster than deacylation for both ACh and ASCh, k2/k3 = 1.8 and 2.3, respectively [21]. Values for (+)acetyl-β-methylcholine/(+)acetyl-β-methylthiocholine show that acylation is the rate-limiting step for hydrolysis of the oxo-ester (k2,o/k3 < 0.11) whatever the value of β (Fig. 3C). This situation very likely results from the steric effect of the β-methyl group that impairs the specific electronic and steric contributions of the ethereal oxygen atom. For the thio-ester the calculation leads to an incon-

clusive result (see Fig. 3A). However, owing to the larger size of S compared to O, it is likely that the electronic and steric contributions of the hetero-atom will be more pronounced for the thio-ester than for the oxo-ester. Thus, acylation is very likely the rate-limiting step for acetyl-β-methylthiocholine too. This would happen with 0.053 < β < 0.1. Calculations for ScdCh/ScdSCh (a = 1.7) again give inconclusive results (Fig. 3D, for ScdCh). However, the asymptotic value of k2,s/k3 = 1.4, and the β1 value of 5.7 for k2,s/k3 = 1 suggest that acylation is the rate-limiting the step or that acylation and deacylation are partly rate-limiting for hydrolysis of the thio-ester. Calculations of k2/k3 from measured values of kcat, Km and Ks for the bulkiest pair of substrates, (−)cocaine/(−)thiococaine [63,64], show that acylation is rate-limiting for both substrates (Table 4). This confirms the trend that steric effects of the bulky alcohol and acyl moieties counteract the influence of the ethereal atom on the acylation rate. Comparison of kcat for hydrolysis of oxo-esters to that for thio-esters, by wild-type BuChE, indicates that introduction of the thio can cause the acylation step to be favored or disfavored. The ratio k2/k3 reflects the difference. For a given pair of substrates, k2/k3 depends on the structure of both the alcohol and acyl moieties. The effect of structure can offset the electronic and steric effects of substituting the ethereal O-atom with S. The effect of structure can be translated into different conformational adjustments between substrate and the active site of the enzyme, which occur on the path toward the transition state structure (ES‡) that leads to formation of the acyl intermediate (EA) [24,65]. Stabilization of transition state by conformational distortions implies that the enzyme active site is conformationally plastic. The hysteretic induction period observed with certain substrates reflects this plasticity. BuChE in solution is composed of two populated conformations, E and E', in slow equilibrium; certain substrates select one preexisting forms, shifting the equilibrium toward that conformer; steady-state velocity is reached when all enzymes molecules are in this active state [22]. 3.5. Molecular docking Molecular docking of BzCh/BzSCh and PhA/PhSA in wildtype BuChE was performed in order to verify whether the positions/orientations of these substrates in the ES complex could explain the differences in acylation rate. As previously shown [23] BzCh and BzSCh bind in a similar productive mode (Fig. 5A): the choline quaternary ammonium interacts with W82, the benzoyl moiety fits in the acyl-binding pocket, the carbonyl oxygen makes hydrogen bond contact with residues of the oxyanion hole (Table 5), and the carbonyl carbon is within bonding distance of the catalytic serine 198. Interestingly, the lowest energy complexes for binding of PhA and PhSA to the active site of wild-type BuChE are similar to those for BuCh and BuSCh, but their positions are not productive in that the catalytic serine (198) is too far from the carbonyl carbon to affect catalysis (Fig. 5B). In this conformation, the phenyl ring interacts with W82, and the

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Fig. 5. Views of energy-minimized complexes of ester and thio-ester homologues in the active site of wild-type BuChE (pdb code 1POM). Panel A is the productive complex for BzCh and BzSCh. Panel B is the non-productive complex for PhA and PhSA (carbonyl oxygen of substrate is H-bonded to W430 and Y440). Panel C is the lowest-energy productive complex for PhA with phenyl ring in the acyl-binding pocket. Panel D is the lowest-energy productive complex for PhA with methyl in the acyl-binding pocket. Substrates are represented in balls and sticks. Carbon atoms of oxo-esters (BzCh and PhA) are in light grey, while those of thio-esters (BzSCh and PhSA) are in black. Labeled residues are: D70 and Y332, the peripheral anionic site; W82, the π-cation binding site that interacts with the trimethyl ammonium head of BzCh and BzSCh in panel A; S198 and H438, components of the catalytic triad; E197, part of substrate binding sub-site and stabilizer of tetrahedral intermediates; G116, G117 and A199, the oxyanion hole residues; W231, component of the acyl-binding pocket along with residues L286 and V288 (not shown). See Table 5 for interatomic distances.

carbonyl oxygen is hydrogen bonded to 3 potential donors: Y440 and the antiparallel indole rings of W82 and W430 (Table 5). Productive complexes with higher binding energy were observed among the 100 runs of Autodock calculation. In these cases, the carbonyl oxygen makes hydrogen bond contact with the oxyanion hole, and either the phenyl ring or the methyl group fits in the acyl-binding pocket (Table 5, Fig. 5C, and D). Because of the moderate size of PhA or PhSA and the large available volume of the gorge, it is possible that a second molecule of substrate binds in a productive way when one molecule is already bound in the non-productive way. Nonproductive binding could be as shown in Fig. 5B. The binding of 2 substrate molecules could be synergistic and the nonproductive molecule could be present during the full catalytic

cycle. In this case, the affinity of substrate for the nonproductive binding site would be higher than the affinity for the productive binding site, and the observed Km would actually be an apparent parameter. Alternatively, with certain mutants, the substrate molecule could bind in the non-productive way first, and then equilibrate into a productive complex with low activity. Then substrate activation (Table 2) could be explained by binding of a second substrate into the active site gorge such that there is a substrate in both the productive and nonproductive positions. Molecular docking of BzCh/BzSCh and PhA/PhSA in modelled mutants was also performed. The binding properties were so dramatically altered that Autodock was unable to find a productive complex for any mutant with any of these substrates. In the worst case (W82A mutant), the complex with the lowest

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Table 5 Interatomic distance (d/Å) between key active site residues and Substrates (S; BzCh, BzSCh, PhA, PhSA) docked into the active site of BuChE in a productive (P) or non productive orientation (NP) BzCh BzSCh PhA

Ocarbonyl(S)–N(Gly116) Ocarbonyl(S)–N(Gly117) Ocarbonyl(S)–N(Ala199) Ocarbonyl(S)–Nε?(Trp82) Ocarbonyl(S)–Nε?(Trp430) Ocarbonyl(S)–O(Tyr440) Oester(S)–Nε2(His438) Sthioster(S)–Nε2(His438) Ccarbonyl(S)–Oγ(Ser198)

2.81 2.73 3.21 – – – 2.95 – 2.76

3.05 2.78 3.29 – – – – 3.29 2.88

PhSA

NP

PMet PPhe NP

– – – 2.73 2.72 2.66 – – –

2.98 2.84 3.36 – – – 2.97 – 3.05

2.71 2.65 3.20 – – – 3.58 – 2.91

– – – 2.70 2.74 2.66 – – –

PMet PPhe 2.71 2.69 3.16 – – – – 3.14 2.91

2.62 2.72 3.11 – – – – 4.44 2.97

PhA and PhSA can orient in two productive conformations with either the Phenyl ring (PPhe) or the methyl group (PMet) in the acyl-binding pocket.

binding energy did not correspond to substrate binding inside the gorge. However, Autodock, being designed for docking simulations of flexible ligands into rigid receptors, cannot take into account the enzyme conformational changes that certainly allow substrate molecules to fit in a productive way into the gorge of these mutants. 4. Discussion 4.1. Effect of ethereal atom substitution on spontaneous hydrolysis of substrates The rate constant of spontaneous hydrolysis of substrates, kuncat in Table 4, depends on the concentration of hydroxide ions [OH−], ([OH−] = 10− 7 M at pH = 7.0), and the second order rate constant kII, (kuncat = [OH−]kII). Table 4 shows kuncat data for several oxo-esters. Though no data are available for homologous thio-esters, it was reported that the free energy of hydrolysis of thio-esters in aqueous solution is 2–8 kcal/mol greater than for oxo-esters [69]. Thus, substitution of the ethereal oxygen for sulfur reduces the chemical stability, and the spontaneous hydrolysis of thio-esters is faster than that of corresponding oxoesters at pH 7.0 [34]. Computational studies also indicate that oxo-esters are more thermodynamically stable than thio-esters [69]. That fits with the Pauling electronegativity, χp, which is 3.44 and 2.58 respectively for O and S. As a consequence, the energy needed to break an ethereal bond –O–C is about 350 kJ/ mol while it is only 260 kJ/mol for an –S–C bond. Thio-esters are therefore more reactive than oxo-esters. 4.2. Effect of ethereal atom substitution on BuChE-catalyzed hydrolysis of substrates Most oxo-esters are hydrolyzed by BuChE at faster rates than their homologous thio compounds. This indicates that the intrinsic electronegativity of the ethereal atom is balanced by other contributions. These contributions are obviously dependent on the reactive pre-organized architecture of the enzyme active center and other physico-chemical properties of the

oxygen atom compared to the sulfur atom. The following factors might be considered in this regard. (1) The bigger size of S compared to O, 0.18 nm against 0.14 nm for van der Waals radius, would be expected to affect the position of the carbonyl carbon in the active site. Because the active site was designed to accommodate oxygen in the etheral position, substitution of sulfur would be expected to move the carbonyl carbon away from its optimal location, thereby reducing the effectiveness of acylation. Substitution of the even larger Se for O was found to cause a more pronounced effect on the acylation rate in electric eel AChE catalysis, with a decrease in k2/k3 from oxo-ester, to thio-ester, and then to seleno-ester [70]. (2) The N+ –C–C–O (Ψ2) torsion angle has a gauche conformation for acetylcholine in solution [71] while it is trans for ASCh and ASeCh [72–74]. The trans conformation is the preferred conformation for binding to the active site, therefore thio-esters would be favored over oxo-esters. Conformational preference will primarily affect the binding step of substrate (Ks), e.g., Ks of BzCh and BzSCh for wild-type BuChE are 14.1 and 3.3 μM, respectively [22]. (3) In addition, the oxo-ether bond provides greater freedom of rotation and less steric hindrance to the carbonyl group. Thus, oxo-esters may adopt more conformations than their homologous thio-esters. This is particularly true for acetylcholine compared to acetylthiocholine, and it is even more pronounced for acetyl-selenocholine. Indeed, the surface of conformational energy maps for energetically favorable torsional angles Ψ1 and Ψ2 shrinks from acetylcholine to acetylselenocholine [73,74]. Thus, the conformational preference leads to decrease in Ks (O > S > Se), but alters the acylation rate. However, by virtue of Eq. (5), the overall effect can be an increase in catalytic efficiency (kcat/Km) from oxo-to seleno-esters [70]. (4) Study of acetylcholine analogs showed marked differences in the electron distribution around the ethereal hetero-atom between oxo-, thio- and selenoesters. This affects the electrostatic potential of the carbonyl oxygen as follows: oxo-esters are more electronegative than thioesters that are more electronegative than selenoesters [72]. Since H-bonding to the carbonyl oxygen is a critical feature of the active site, decreased electronegativity in the carbonyl oxygen would be expected to contribute to a reduction in the reactivity of cholinesterases toward the thio- and seleno-esters. (5) The capability of making H-bonds at the etheral atom is another important feature. Dipole moment measurements showed that the ability of the S atom to H-bond is reduced compared to the O atom in isologous esters [75]. The tendency to H-bond ranks as follows: O > S > Se. Thus, Hbonding of the ethereal hetero-atom may exert an influence on binding (ES formation) or for the proper adjustment of the substrate in the acylation transition state (ES≠ formation). However, the leaving groups of oxo-/

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thio-/seleno-esters are of decreasing basicity at the ethereal atom in the order O > S > Se, so that the Hbonding strength and/or proton transfer to the ethereal atom in the acylation transition state are of decreasing importance to catalysis in the order O > S > Se. Moreover, the rate-limiting step in acylation is likely the formation of the tetrahedral intermediate, so the relative H-bonding capacity of the leaving group should have little effect on the acylation rate. This conclusion is supported by docking experiments with BzCh and BzSCh in the active center of wild-type BuChE, which showed no evidence for H-bonding of the ethereal atom with active site residues in the enzyme–substrate complex, except a possible weak interaction with H438 (cf. ref [23] and present work). In addition, semi-empirical calculations indicated that no significant H-bonding occurred between the ethereal O atom of BzCh and active site groups in the tetrahedral intermediate [76]. These steric, electronic and bonding contributions can combine to make oxo-ester catalysis better than thio-ester catalysis (synergistic effect), or conversely they can combine to make oxo-ester catalysis worse than thio-ester catalysis (antagonistic effect). Or the contributions can balance each other leading to a neutral effect. These three possibilities are illustrated:

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terases than their oxo-analogs (organophosphates) [78]. The reactivity ratio can be of several orders of magnitude, depending on the structure and configuration of the thiolo leaving group. The highest reactivity is obtained with branched thiocholine groups [68]. A thorough comparative molecular dynamic analysis of the orientation of carboxyl- and phosphonyl-esters in the active center of AChE demonstrated the importance of absolute stereochemical configurations of substrates for reactivity and enantiomeric selectivity of cholinesterases [79]. (c) In the case of BzCh/BzSCh the effects are synergistic. Given that there is no significant change in geometry of the active center of BuChE between the planar ES complexes (sp2 geometry of the substrate) and the tetrahedral intermediate of acylation complex (sp 3 geometry of the activated substrate S≠) [2,80], substitution of the ethereal oxygen, on one solid angle of the tetrahedron, by S or Se atoms is expected to cause distortion in the tetrahedral geometry. This effect appears to be more pronounced when bulky P1 moieties interact with W82 and other residues in the π-cation binding site. k2 ratios for BzCh/BzSCh and (−)cocaine/(−)thiococaine are almost 4-fold different. This suggests that the size of P1 primarily influences absolute values of kinetic constants and the rate-limiting step (Table 4). 4.3. Role of the oxyanion role

(a) In the case of PhA/PhSA, present results show that the atom substitution has a neutral effect. Inspection of results previously reported for short-chain alky-choline and thiocholine esters leads to the same conclusion. In particular, turnover ratios (kcat,O/kcat,S) for acetyl-, propionyl-, and butyryl-choline versus their homologous thiocholine esters are close to one (Table 4). This suggests that for each pair of substrates, acylation rate constants may not be very different or that deacylation is rate limiting. Kinetic analysis of oxo-/thio-choline-ester series of higher rank should allow us to decide between these alternative hypotheses. (b) Antagonistic contributions can explain why certain oxoesters are not hydrolyzed (or are poorly hydrolyzed) by wild type BuChE compared to their thio-ester counterparts. This is the case of acetyl-β-methylcholine and acetyl-β-methylthiocholine (Table 4). The L(+)oxo-ester is hydrolyzed by BuChE at a low rate (10% of the Vmax of BzCh [77] while the L(+) thio-ester is hydrolyzed at a rate similar to that of BzCh [34]. This has been explained by a shielding effect of the β-methyl group that prevents Hbonding of the ethereal O atom with the catalytic histidine [34]. The D(−) stereoisomers are not hydrolyzed by either BuChE or AChE [77]. Results with (±)AβMCh/(±) AβMSCh, as well as data for cocaine isomers (Table 4), emphasize the importance of enantioselectivity in cholinesterase reactivity. Similar antagonistic effects can also explain high reactivity of phosphorothiolate. i.e., organophosphyl esters in which the leaving group is P–S bonded. These phosphylesters are more reactive with cholines-

X-ray diffraction, mutagenesis studies, and the use of transition-state analogs indicate that tetrahedral intermediates formed during cholinesterase-catalyzed reactions are stabilized by H-bonds with the oxyanion form of the carbonyl oxygen [38,39]. This conclusion is supported by computational chemistry calculations [10,40,41,79,81]. Electrophilicity of the carbonyl carbon is correlated to electronegativity of the carbonyl oxygen atom [72]. But the stereoelectronic control of the carbonyl oxygen binding in the oxyanion hole is influenced by the structures of P1 (the alcoholic component of the ester) and P2 (the acyl component of the ester) and by the nature of the ethereal hetero-atom. For example, substitution of ethereal oxygen by sulfur and selenium was found to decrease the apparent bimolecular rate constant for carbamylation of AChE by carbaryl derivatives [82]. The crucial role of the oxyanion hole in H-bond stabilization of tetrahedral intermediates is exemplified by the absence of reactivity of cholinesterases towards thiono-esters (C_S) where sulfur substitutes for the carbonyl oxygen. Similarly, thiono-organophosphates (P_S) are very poor phosphorylating agents of cholinesterases [68] compared to oxono analogs (P_O), e.g., parathion versus paraoxon. And, thiono-carbamates do not react even though they can reversibly bind to the active center [82]. The fact that the ethereal hetero-atom affects the adjustment and H-bonding of the carbonyl oxygen in the oxyanion hole has an important consequence on the acylation reaction. If, Hbonding of oxo-ester tetrahedral intermediates to oxyanion is stronger than for thio-esters, then acylation transition states of oxo-esters are much better stabilized. This lowers the reaction

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barrier for acylation with oxo-esters. Conversely, formation of the acylation tetrahedral intermediate of thio-esters is disfavored. This is the situation where a > 1 (Table 3). Moreover, previous study on the effects of high pressure on BuChEcatalyzed hydrolysis of BzCh and BzSCh, showed that the enzyme–substrate complementarity is better with BzSCh than with BzCh. This suggested that higher activation energy is needed for the ES complex with BzSCh to jump over the acylation transition state barrier [23]. Lastly, due to impairment in tetrahedral formation and transition state stabilization, mutations in the oxyanion hole and vicinal loci can lead to significant decreases in activity (cf. Ref. [17] for organophosphate hydrolysis activity of corresponding mutants) and in binding affinity (cf. Table 1 for BzCh vs. BzSCh). Therefore, one would expect oxo-esters to be better substrates than thioester. However, with certain pairs of substrates (PhA/PhSA and L(+)AβMCh/ L(+)AβMSCh, where a < 1) the thio-ester is the best substrate. It is likely that this results from chemical features of those substrates that provide a better adjustment of the acylation transition state of the thio-ester in the oxyanion hole. Acknowledgments This work was supported by DGA/DSA/03co010-05/PEA 01 08 07 and EMA/LR 2006 to PM and OL and, US Army Medical Research and Materiel Command DAMD 17-01-10776 to OL. The authors thank William I. Goodrich (UNMC, Eppley Institute) for his expert assistance in computer science. References [1] J. Massoulié, L. Pezzementi, S. Bon, E. Krejci, F.M. Vallette, molecular and cellular biology of cholinesterases, Prog. Neurobiol. 41 (1993) 31–91. [2] Y. Nicolet, O. Lockridge, P. Masson, J.C. Fontecilla-Camps, F. Nachon, Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products, J. Biol. Chem. 278 (2003) 41141–41147. [3] S. Darvesh, D.A. Hopkins, C. Geula, Neurobiology of butyrylcholinesterase, Nat. Rev., Neurosci. 4 (2003) 131–138. [4] A.L. Guillozet, J.F. Smiley, D.C. Mash, M.M. Mesulam, Butyrylcholinesterase in life cycle of amyloid plaques, Ann. Neurol. 42 (1997) 909–918. [5] E. Giacobini, Cholinesterases: new roles in brain function and in Alzheimers's disease, Neurochem. Res. 28 (2003) 515–522. [6] M.M. Mesulam, A. Guillozet, P. Shaw, A. Levey, E.G. Duysen, O. Lockridge, Acetylcholinesterase knockouts establish central cholinergic pathway and can use butyrylcholinesterase to hydrolyze acetylcholine, Neuroscience 110 (2003) 627–639. [7] O. Lockridge, Genetic variants of human serum butyrylcholinesterase influence the metabolism of the muscle relaxant succinylcholine, Pharmac. Ther. 47 (1990) 35–60. [8] G.F. Gilmer, L.M. Moriarty, M.N. Lally, J.M. Clancy, Isosorbide-based aspirin prodrugs. II. Hydrolysis kinetics of isorsorbide diaspirinate, Eur. J. Pharm. Sci. 16 (2002) 297–304. [9] Z. Kovarik, V. Simeon-Rudolf, Interaction of human butyrylcholinesterase variants with bambuterol and terbutaline, J. Enz. Inhib. Med. Chem. 19 (2004) 113–117. [10] Y. Pan, D. Gao, W. Yang, H. Cho, G. Yang, H.-H. Tai, C.-G. Zhan, Computational redesign of human butyrylcholinesterase for anticocaine medication, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 16656–16661. [11] A.R. Main, Mode of action of anticholinesterases, Pharmac. Ther. 6 (1979) 579–628. [12] T.C. Marrs, Organophosphate poisoning, Pharmac. Ther. 58 (1993) 51–66.

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