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Phosphobutyrylcholinesterase: Phosphorylation of the Esteratic Site of Butyrylcholinesterase by Ethephon [(2-Chloroethyl)phosphonic Acid] Dianion J. Eric Haux, Gary B. Quistad, and John E. Casida* Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 114 Wellman Hall, University of California, Berkeley, California 94720-3112 Received February 11, 2000
Ethephon [(2-chloroethyl)phosphonic acid] has two seemingly unrelated types of biological activity. It is a major agrochemical absorbed by crops, slowly releasing ethylene as a plant growth regulator. Ethephon also inhibits the activity of plasma butyrylcholinesterase (BuChE) in humans, dogs, rats, and mice. This is totally unexpected for an ionized phosphonic acid (mostly the dianion at physiological pH), in contrast to the classical inhibitors (nonionized triester phosphates) which phosphorylate serine at the active site. This study tests the hypothesis that ethephon (as the dianion) also acts as a phosphorylating agent in inhibiting BuChE activity. The sensitivity of plasma BuChE to ethephon (90 min preincubation at 25 °C) is greatest for humans, dogs, and mice (IC50 ) 6-23 µM), intermediate for chickens, rabbits, rats, and guinea pigs (IC50 ) 26-53 µM), and lowest for pigs and horses (IC50 ) 92-172 µM). The IC50 decreases linearly with time on a log-log scale to values of 0.15-0.3 µM for human, dog, and horse BuChE at 24 h. The inhibition rate is generally related to ethephon concentration, consistent with a bimolecular reaction, e.g., phosphorylation. The extent of inhibition of the esteratic activity of BuChE by ethephon is directly proportional to the extent of inhibition of [3H]diisopropyl phosphorofluoridate ([3H]DFP) postlabeling which is not reversible on removing the ethephon, either directly or after further incubation for 24 h at 25 °C. These observations strongly suggest that ethephon, as DFP, phosphorylates human plasma BuChE at Ser-198 of the esteratic site, or more generally, the formation of a phosphobutyrylcholinesterase. With human plasma BuChE, (2-bromoethyl)- and (2-iodoethyl)phosphonic acids have lower affinities for the site than ethephon but higher phosphorylation rate constants, consistent with their relative hydrolysis rates at pH 7.4 (phosphorylation of water). (2Chlorohexyl)phosphonic acid is a poor inhibitor, perhaps being too reactive with water. Thus, potency differences for ethephon and its analogues with BuChE of various species depend on both the affinities and phosphorylation rates, i.e., the binding and reactivity of the (2-haloalkyl)phosphonic acid dianion in the esteratic site.
Introduction Ethephon [(2-chloroethyl)phosphonic acid] inhibition of plasma butyrylcholinesterase (BuChE)1 activity is an intriguing anomaly to the well-defined reaction of O,Odialkyl phosphates with BuChE and acetylcholinesterase (AChE), leading to dialkyl phosphorylation of the activesite serine. This major agrochemical is used as a plant growth regulator, penetrating into the tissue and decomposing to ethylene which affects growth, flowering, fruit maturation, and other processes (1-3). The secondary effect of BuChE inhibition was originally observed in vitro and in vivo at high levels in rats and mice (4, 5) and more recently extended to low levels in subacute and chronic studies with beagle dogs and human subjects (6). * To whom correspondence should be addressed: Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 115 Wellman Hall, University of California, Berkeley, CA 94720-3112. Telephone: (510) 642-5424. Fax: (510) 642-6497. E-mail:
[email protected]. 1 Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; BuTCh, butyrylthiocholine; DFP, diisopropyl phosphorofluoridate; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); IC50, median inhibitory concentration; OP, organophosphate.
These findings are totally unexpected and therefore challenging to explain mechanistically and interpret toxicologically. The mechanism by which ethephon inhibits BuChE may be related to the observations of Maynard and Swan (7) and Segall and co-workers (8) that the diionized form can serve as a phosphorylating agent. Ethephon liberates phosphate and ethylene in aqueous media and phosphorylates primary alcohols in the presence of water (8). The BuChE inhibition reaction is relatively slow with more inhibition in vitro at 90 min than at 15 min and 25 °C (9), and it is selective for BuChE compared with AChE (4, 5). (2-Bromoethyl)phosphonic acid is a better inhibitor than ethephon and is also more reactive as a phosphorylating agent (9). These observations led to the proposal that “the esteratic site of the enzyme is phosphorylated, perhaps at the serine residue, forming an aged phosphoenzyme” (9). This proposal is analogous to the dialkyl phosphorylation reaction by O,O-dialkyl phosphates [e.g., diisopropyl phosphorofluoridate (DFP)] but is mechanistically quite distinct since it requires that a phosphonic acid anion selectively bind to BuChE and react with serine at the active site.
10.1021/tx000027w CCC: $19.00 © 2000 American Chemical Society Published on Web 06/17/2000
Phosphobutyrylcholinesterase from Ethephon
Chem. Res. Toxicol., Vol. 13, No. 7, 2000 647 haloalkyl analogues according to the following equations (13). k+1
BuChE + ethephon y\ z k -1
k+2
[BuChE‚ethephon] 98 BuChE-OP(O)(OH)2 Ka )
Figure 1. Phosphorylation of human plasma BuChE by ethephon and [3H]DFP and use of [3H]DFP postlabeling to establish Ser-198 as the position of ethephon phosphorylation.
This study tests in three ways the hypothesis that ethephon inhibition of BuChE involves phosphorylation of the active-site serine (Figure 1). First, the potency of ethephon is defined with plasma of nine species, and the inhibition rate is related to a bimolecular phosphorylation mechanism. Second, the potential reaction with the active-site serine is examined as inhibition of both esteratic activity and postlabeling by [3H]DFP. Third, three (2-haloalkyl)phosphonic acids are compared with (2-chloroethyl)phosphonic acid to establish a possible relationship between rates of hydrolysis and BuChE inhibition. The findings are finally related to the toxicological aspects and biological relevance of ethephoninduced phosphoprotein formation.
Materials and Methods Chemicals and Plasma. Sources of compounds were as follows: ethephon (98% pure) from Chem Service (West Chester, PA), butyrylthiocholine iodide (BuTCh), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), purified horse plasma BuChE, and other plasma (lyophilized) from Sigma (St. Louis, MO), and purified human plasma BuChE (lyophilized) from Lee Scientific (St. Louis, MO). Methods used to prepare (2-haloalkyl)phosphonic acids were as follows: that of Dreef et al. (10) for (2-bromoethyl)phosphonic acid and that of Gregory and Higgins (11) for (2iodoethyl)phosphonic acid and (2-chlorohexyl)phosphonic acid. BuChE Activity Assays. The activity of BuChE was determined as the level of BuTCh hydrolysis, by monitoring thiocholine liberation with the DTNB reagent (12). Reactions were carried out in Costar cell-culture 96-microwell plates at 25 °C in 100 mM phosphate buffer (pH 7.4) (200 µL volume) containing the enzyme (lyophilized plasma or purified BuChE as specified) and DTNB (0.3 mM final concentration). BuTCh (0.5 mM final concentration) was added in 50 µL of buffer to determine BuChE activity. The absorbance change at 25 °C was monitored with the UVmax Plate Reader (Molecular Devices, Sunnyvale, CA) at 405 nm in 15 s intervals for 15 min, and the data were collected with SoftMax software and graphically displayed with SigmaPlot software. Eight replicates were run in each experiment. With each BuChE source, the absorbance change in the absence of inhibitor was directly proportional to the enzyme level. BuChE Inhibition Studies. The candidate inhibitor was incubated with BuChE and DTNB for 15 min to 24 h prior to addition of BuTCh for residual activity assay as described above. The BuChE activity in controls changed negligibly over a period of 24 h, but in any case, controls were always run to determine possible activity loss unrelated to inhibitor action. Candidate inhibitors were added in buffer in which they were completely soluble. Unless specified otherwise, the series of 1, 3, 10, 30 µM, etc., in the concentration range yielding 10-90% inhibition was used in determining the median inhibitory concentration (IC50) as a function of time. Apparent Dissociation, Phosphorylation, and Bimolecular Rate Constants. Affinity and rate constants are calculated for the inhibition of BuChE by ethephon and its
k-1 + k+2 k+1
Enzyme phosphorylation generally follows bimolecular kinetics analyzed by plotting log % activity versus time. Pseudo-firstorder rate constants (k) are calculated for each inhibitor concentration ([inhibitor]), where the slope equals -k/2.303. The constants for apparent dissociation (Ka), phosphorylation rate (k+2), and bimolecular rate (ka) are calculated from a plot of [inhibitor]/k versus [inhibitor], where the slope equals 1/k+2, the y-intercept equals Ka/k+2, and ka ) k+2/Ka. Relation between Inhibition by Ethephon of BuChE Activity and [3H]DFP Postlabeling. The goal was to use a standard incubation mixture for purified human BuChE with ethephon at various concentrations and after 90 min at 25 °C to determine the residual BuChE activity and the portion of the BuChE active site still available for labeling with [3H]DFP. Ethephon solutions in 100 mM phosphate buffer (pH 7.4) (150 µL) at 25 °C were added to the 96-microwell plate with one concentration for three wells. DTNB (1.5 mM) and BuChE (0.19 unit, 15 µg of protein) in 50 µL of the phosphate buffer were then added to each well. After 90 min, BuTCh (50 µL of a 2.5 mM solution) was added to each well and the plate read at 405 nm for 15 min at 15 s intervals. This portion of the experiment established the curve for inhibition of BuChE activity. For [3H]DFP postlabeling, reactions identical to those described above were carried out in 0.5 mL snap-cap tubes. Instead of BuTCh, [3H]DFP (NEN Life Science Products, Boston, MA) (8.4 Ci/mmol) [1 × 107 dpm in 50 µL of 100 mM phosphate buffer (pH 7.4)] was added to each tube, and the tubes were incubated for 15 min at 25 °C. Laemmli sample buffer (14) (modified by increasing the glycerol content to 37.5%) (50 µL) was added to each tube. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on a Mini-PROTEAN II system (BioRad, Hercules, CA) using 8% running and 5% stacking gels according to the method of Laemmli (14) and Sambrook et al. (15). Protein markers (including 66 and 110 kDa) were loaded on each lane followed by half of the sample (150 µL) for each of two gels. After being stained with Coomassie Brilliant Blue R-250, the gels were destained and incubated in 5% glycerol for 1-2 h at 25 °C. The 120-60 kDa region (evident from the marker proteins) of each sample lane was transferred to a scintillation vial; 30% hydrogen peroxide (0.5 mL) was added, and the samples were incubated at 60 °C for 3 h to melt the gel. Once the samples had cooled to room temperature, 1 M HCl (100 µL) and Hionicfluor scintillation fluid (Packard, Meriden, CT) (10 mL) were added. A lane on each gel with the marker proteins (but no labeled sample) was used as background. Control labeling (no ethephon) was 4000-7000 dpm in different experiments. The extent of inhibition of labeling by ethephon was expressed relative to the extent of labeling with no ethephon in the same experiment. The [3H]DFP postlabeling reaction was further characterized by treating purified human BuChE as described above with 0 or 256 µM ethephon for 24 h, then removing the ethephon, and recovering the BuChE by size-exclusion chromatography (16). The recovered enzyme, either immediately or after 24 h at 25 °C, was subjected to the [3H]DFP postlabeling procedure with analysis of the labeled enzyme by size-exclusion chromatography.
Results Species Differences in Plasma BuChE Activity (Table 1). Lyophilized plasma from nine species and
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Haux et al.
Table 1. Species Differences in Plasma BuChE Activity and Sensitivity to Ethephon speciesa dog human plasma purified mouse chicken rabbit rat guinea pig pig horse plasma purified
protein level activityb ( SD per assay (µg) (mAU)
IC50 ( SDc (µM)
0.54 ( 0.02
6 ( 2 (12 ( 6)d
80e 0.33 74e 63 52e 77e 71 72e
1.1 ( 0.1 60 ( 2 0.85 ( 0.03 0.32 ( 0.02 0.089 ( 0.027 0.14 ( 0.01 1.1 ( 0.1 0.044 ( 0.018
11 ( 6 (23 ( 9)d 21 ( 2 13 ( 3 26 ( 4 38 ( 1 39 ( 3 53 ( 4 92 ( 3
80e 0.0044
1.1 ( 0.1 17500 ( 650
71
166 ( 2 (172 ( 25)d 267 ( 2
a Lyophilized plasma or purified BuChE. b Activity as milliabsorbance units per microgram of protein per minute at 25 °C (n ) 8). BuTCh (0.5 mM final concentration) hydrolysis by plasma (or purified BuChE at a specified protein level) in 100 mM sodium phosphate buffer (pH 7.4, 250 µL). The activity of goat and bovine plasma was too low for analysis ( iodoethyl or bromoethyl > chloroethyl > fluoroethyl (this study and refs 7, 9, and 11). BuChE inhibition by this series depends on both the binding affinity and the phosphorylation rate. The fluoroethyl compound is not an inhibitor (9). The apparent affinity for BuChE decreases in the following order: chloroethyl > bromoethyl > iodoethyl > 2-chlorohexyl; the phosphorylation rate decreases in the following order: bromoethyl ) iodoethyl > chloroethyl ) 2-chlorohexyl. The rate and extent of inhibition by each (2-haloalkyl)phosphonic acid are therefore a balance of affinity and phosphorylation rates at the esteratic site and the stability of the compound. The phosphorylation of BuChE by ethephon may involve a pentavalent transition state intermediate such as that shown in Figure 7 in which the negatively charged oxygen is stabilized by hydrogen bonding to the main chain nitrogens in the oxyanion hole (18, 21). Perhaps the haloalkyl substituent helps orient the phosphonic acid moiety for favorable reaction; a similar observation has been made for alkyl- and haloalkylphosphonic acid triesters as inhibitors of neuropathy target esterase (22). Toxicological Aspects of Plasma BuChE Inhibition by Ethephon. Ethephon inhibits plasma BuChE in vivo in rats, mice, dogs, and humans (4-6). The in vitro investigation presented here indicates that dog plasma BuChE is most sensitive to ethephon-induced inhibition, that human and mouse BuChE are intermediate, and that rat BuChE is the least sensitive in 90 min assays. The dog therefore appears to be the best predictor model for humans. Plasma BuChE inhibition is a common feature for OP triester insecticides or herbicides (e.g., tribufos) (23) but is unique for ethephon as a
Phosphobutyrylcholinesterase from Ethephon
phosphonic acid plant growth regulator. Ethephon sensitizes mice to succinyldicholine toxicity that can be attributed to BuChE inhibition, but this requires a high dose relative to many OP triesters (24). The in vivo inhibition of plasma BuChE in mice is partially reversible within 24 h (24), possibly via hydrolysis of the phosphoenzyme. BuChE in vitro is 10-fold more sensitive to inhibition by ethephon than AChE from rat brain (5). AChE from electric eel and housefly head is also inhibited in vitro by ethephon (IC50 ) 410 and 720 µM, respectively, with a 90 min preincubation at 25 °C) (data not shown), but other serine hydrolases (carboxylesterase, elastase, and thrombin) are not affected (IC50 > 2000 µM) (data not shown). Biological Importance of Phosphoproteins. Protein phosphorylation and dephosphorylation by protein kinases and protein phosphatases, respectively, are critical regulatory processes for many cellular functions. Ethephon is one of the few synthetic compounds and perhaps the only agrochemical that spontaneously reacts to generate phosphoprotein(s), altering their biological activity. It is a much better inhibitor of BuChE than AChE (5), in contrast to phosphorus oxychloride that is more potent with AChE (16). On a more general basis, BuChE is the only enzyme known to be inhibited both in vitro and in vivo by ethephon, but it is the only one considered in any detail, and others, if inhibited, would not have been detected in the studies reported. These findings suggest the possibility of controlling the levels of specific phosphoproteins not only by altering the activities of protein kinases and protein phosphatases but also with suitable synthetic phosphorylating agents.
Acknowledgment. The project described was supported by Grant R01 ES08762 from the National Institute of Environmental Health Sciences (NIEHS), NIH, and its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH. It also received support from the University of California Toxic Substances Research and Teaching Program. The ethephon analogues were synthesized by Yoshihisa Tsukamoto and their hydrolysis rates determined by Nanjing Zhang, both in the Berkeley laboratory.
Chem. Res. Toxicol., Vol. 13, No. 7, 2000 651
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