Interaction of the serine hydrolase KIAA1363 with organophosphorus agents: Evaluation of potency and kinetics

Article abstract of DOI:10.1016/j.abb.2015.11.034

Oxons are bioactive metabolites of organophosphorus insecticides (OPs) that covalently inactivate serine hydrolases. KIAA1363 is one of the most abundant serine hydrolases in mouse brain. Although the physiological consequences related to the inhibition o

Full text of DOI:10.1016/j.abb.2015.11.034

Archives of Biochemistry and Biophysics 590 (2016) 72e81
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Interaction of the serine hydrolase KIAA1363 with organophosphorus
agents: Evaluation of potency and kinetics
,
b
,
, b
Matthew K. Ross a *, Kim Pluta a, Victoria Bittles a, Abdolsamad Borazjani a
,
, b, **
J. Allen Crow a
a Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, United States
b Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxons are bioactive metabolites of organophosphorus insecticides (OPs) that covalently inactivate serine
hydrolases. KIAA1363 is one of the most abundant serine hydrolases in mouse brain. Although the
physiological consequences related to the inhibition of KIAA1363 due to environmental exposures to OPs
are poorly understood, the enzyme was previously shown to have a role in the detoxification of oxons.
Here, we overexpressed human KIAA1363 and CES1 in COS7 cells and compared the potency of inhibition
(IC50s, 15 min) of KIAA1363 and CES1 by chlorpyrifos oxon (CPO), paraoxon (PO), and methyl paraoxon
(MPO). The order of potency was CPO > PO >> MPO for both enzymes. We also determined the bimo-
lecular rate constants (kinact/Ki) for reactions of CPO and PO with KIAA1363 and CES1. KIAA1363 and CES1
Received 12 September 2015
Received in revised form
3 November 2015
Accepted 20 November 2015
Available online 23 November 2015
Keywords:
Carboxylesterase
Oxon
Bimolecular rate constant
Enzyme inhibition
Chlorpyrifos oxon
Paraoxon
were inactivated by CPO at comparable rates (4.4 ꢀ 106 sꢁ1 Mꢁ1 and 6.7 ꢀ 106 sꢁ1
M
ꢁ1, respectively),
whereas PO inactivated both enzymes at slower rates (0.4 ꢀ 106 sꢁ1 Mꢁ1 and 1.5 ꢀ 106 sꢁ1 Mꢁ1
,
respectively). Finally, the reactivation rate of KIAA1363 following inhibition by CPO was evaluated.
Together, the results define the kinetics of inhibition of KIAA1363 by active metabolites of agrochemicals
and indicate that KIAA1363 is highly sensitive to inhibition by these compounds.
© 2015 Elsevier Inc. All rights reserved.
1. Introduction
acute toxicity by inhibiting the activity of acetylcholinesterase, a
member of the serine hydrolase superfamily, resulting in cholin-
Organophosphorus insecticides (OPs) are used in significant
quantities in agriculture to prevent pest infestation. Approxi-
mately 73 million pounds of OP insecticides were used in the
United States in 2001 [1]. Human exposure to these compounds is
widespread; for example, dialkylphosphate metabolites of OP in-
secticides were detected in urine samples of roughly half the
NHANES study population (http://www.cdc.gov/exposurereport/
pdf/FourthReport.pdf). Toxicity of OPs is due to the in vivo gen-
eration of oxons, which are formed mainly in liver via cytochrome
P450-catalyzed oxidation of the parent OP. The oxons exert their
ergic overload [2e4]. The oxons are also capable of reacting with
several other serine hydrolases [5,6]. For example, oxons can
covalently modify the catalytic serine residue in carboxylesterase
1 (CES1), which is a serine hydrolase found in large quantities in
human liver and other tissues. The reaction with CES1 is an
important mechanism by which oxons are detoxified [6]. We have
previously defined the kinetic constants (bimolecular rate con-
stant, kinact/Ki) for the covalent reaction of several oxons and car-
bamates with recombinant CES1 [7], [8]. CES1 was an extremely
effective scavenger of oxons, exhibiting a kinact/Ki of ~107 sꢁ1 Mꢁ1
for chlorpyrifos oxon (CPO). In addition, the phosphorylated
adduct was relatively stable (t1/2 ¼ 31 h at 37 ꢂC) [7]. Thus, CES1
appears to act as a covalent sink that functionally removes these
toxic compounds from the body.
List of Abbreviations: AcMAGE, acetyl monoacylglycerol ether; CES1, carbox-
ylesterase 1; CES2, carboxylesterase 2; CPO, chlorpyrifos oxon; MGL, monoglyceride
lipase; MPO, methyl paraoxon; 4-MUBA, 4-methylumbelliferyl acetate; pNPV, para-
nitrophenyl valerate; OPs, organophosphorus insecticides; PO, paraoxon.
* Corresponding author. P.O. Box 6100, MS 39762, United States.
** Corresponding author. P.O. Box 6100, MS 39762, United States.
E-mail addresses: mross@cvm.msstate.edu (M.K. Ross), crow@cvm.msstate.edu
(J. Allen Crow).
KIAA1363 is
a 45e50-kDa glycosylated enzyme that is
expressed in most tissues, including macrophages [9], and is one
of the most abundant serine hydrolases in mouse brain mem-
branes [10]. The physiological role of KIAA1363 is still not
completely understood, although it is highly expressed in several
http://dx.doi.org/10.1016/j.abb.2015.11.034
0003-9861/© 2015 Elsevier Inc. All rights reserved.

M.K. Ross et al. / Archives of Biochemistry and Biophysics 590 (2016) 72e81
73
invasive cancer cell lines [11]. One biochemical pathway regulated
by KIAA1363 is the removal of an acetyl moiety from the sn-2
hydroxyl group of the glycerol backbone of acetyl mono-
acylglycerol ethers (AcMAGEs) [12]. AcMAGEs are lipid mediators
that contribute to the invasive phenotype of certain cancer cells. In
addition, KIAA1363 was reported to have cholesteryl ester hy-
drolase activity, leading it to be renamed neutral cholesteryl ester
hydrolase (nCEH) [13], although this particular biochemical ac-
tivity and designation for KIAA1363 remains highly controversial
[14,15]. With respect to its toxicological properties, murine
KIAA1363 was shown to have a high affinity for CPO and was the
predominant CPO binding and metabolizing enzyme in the mouse
brain [9,10]. In vitro studies using brain membranes from wildtype
mice showed that 1 nM [3H]-CPO could be completely hydrolyzed
after a 60 min incubation, whereas brain membranes from
KIAA1363-null mice were 13-fold less efficient at hydrolyzing CPO.
Furthermore, on the basis of SDS-PAGE analysis, KIAA1363 was the
most prominent protein in brain membranes radiolabeled by 1 nM
[3H]-CPO [10]. These results implied that KIAA1363 could be
covalently modified by CPO and its enzyme activity transiently
inhibited during the course of the 60-min reaction, but that the
enzyme was reactivated to its functional state by the end of
60 min, i.e. CPO is in effect a slowly turned over substrate.
Importantly, KIAA1363e/e mice were found to be more susceptible
to OP-induced acute intoxication than wildtype mice [16], indi-
cating a protective role for this enzyme. However, whether tran-
sient inhibition of KIAA1363 by long-term exposure to low
concentrations of CPO might cause adverse toxicological effects
due to changes in key lipid metabolism pathways is unclear.
Furthermore, current physiologically-based pharmacokinetic/
pharmacodynamic (PBPK/PD) models of OPs do not incorporate
KIAA1363-dependent detoxication pathways [17]. These are
important gaps that need to be addressed.
In this study, we evaluated the kinetic parameters that describe
the interaction of oxons with KIAA1363. We overexpressed human
KIAA1363 in COS7 cells to examine its sensitivity to OP oxons and
determined the half-maximal inhibitory concentrations (IC50s)
when treated by CPO, paraoxon (PO), and methyl paraoxon (MPO).
These oxons are derived from chlorpyrifos, parathion, and methyl
parathion, respectively, and are common pesticides used in agri-
cultural settings. In addition, we determined the bimolecular rate
constants for the reaction of CPO and PO with KIAA1363, and
compared them to the rate constants obtained with CES1, a highly
efficient enzyme at detoxifying oxons [7]. Finally we evaluated the
reactivation rate constant of KIAA1363 phosphorylated by reaction
with CPO, i.e. the rate constant for the hydrolysis of the KIAA1363-
diethylphosphate adduct.
studying a protein in a cell lysate. Only by examining a specific
reaction using a purified, isolated protein can one determine the
intrinsic kinetic parameters (kcat and Km) of the enzyme. In cell
lysates, these values are “apparent” under the specific conditions
employed.
2. Materials and methods
2.1. Cells, chemicals, reagents, and transfections
COS-7 cells, Dulbecco's Modified Eagle's Medium (DMEM),
gentamicin sulfate solution (50 mg/ml), and Hanks' balanced salt
solution without calcium, magnesium or phenol red were pur-
chased from the American Type Culture Collection (ATCC) (Mana-
ssas, VA). Fetal bovine serum (FBS) was purchased from Invitrogen
(Carlsbad, CA). pCMV6-XL5 expression vectors containing either
human KIAA1363 cDNA [also called arylacetamide deacetylase like
1
(AADACL1) or neutral cholesteryl ester hydrolase (nCEH)]
(REFSEQ accession number: NM_001146276.1) or human CES1
cDNA (isoform c) (REFSEQ accession number: NM_001266.4) were
purchased from Origene (Rockville, MD). KIAA1363 and CES1
plasmids were transfected into COS-7 cells in 60-mm plates using
the manufacturer's instructions (FuGene 6, Invitrogen). Trans-
fections of COS-7 cells with an empty vector were performed in
parallel. After 48 h, the cells were washed with PBS, harvested by
scraping, and lysed in ice-cold 50 mM TriseHCl (pH 7.4) buffer by
sonication. The recombinant proteins in COS-7 cell lysates were
used without further purification.
O,O0-Diethyl 3,5,6-trichloro-2-pyridyl phosphate (chlorpyrifos
oxon) and O,O’-diethyl p-nitrophenyl phosphate (paraoxon) were
kind gifts from Dr. Howard Chambers, Department of Entomology,
Mississippi State University. Oxons were >99% pure when assessed
by thin-layer chromatography [21]. para-Nitrophenyl valerate
(pNPV), 4-methylumbelliferyl acetate (4-MUBA), and all general
reagents and buffers were purchased from Sigma (St. Louis, MO).
The activity-based serine hydrolase probe, fluorophosphonate-
biotin (FP-biotin), was from Toronto Research Chemicals (North
York, Ontario). Avidin-horse radish peroxidase (HRP) was from
Sigma.
2.2. Protein assays
The protein concentration of cell lysates was measured using the
BCA reagent (Pierce, Rockford, IL) according to the manufacturer's
instructions.
2.3. Enzyme assays
The recombinant proteins in COS-7 cell lysates were used
without further purification. The justification for this is as follows.
Fluorescent reporter probes indicate that there is substantial co-
localization of KIAA1363 and endoplasmic reticulum mem-
branes in cells [18], indicating that KIAA1363 is embedded within
this membrane. As compared to cytosolic proteins, integral
membrane proteins are challenging to purify for structural and
functional studies. The KIAA1363 protein that we overexpressed
in COS7 cells also lacked a histidine tag, making its purification
even more challenging. There are advantages to studying a pro-
tein in a cell lysate versus studying the pure protein. Protein ac-
tivity can often be lost when proteins are examined in their
purified, isolated forms [19]. Requisite co-factors, protein part-
ners, and small molecule modulators might be lost when the
protein is purified and studied in isolation. Using cell lysates,
rather than an isolated protein, allows the protein under exami-
nation to be studied in an environment closer to its native
physiological environs [20]. There are, however, disadvantages to
Hydrolysis reactions using the pro-fluorogenic substrate 4-
MUBA (final concentration 250
96-well plate format in a total volume of 300
m
M) were performed at 37 ꢂC in a
mL/well in 50 mM
TriseHCl (adjusted to pH 7.4 at room temperature). COS-7 cell ly-
sates containing overexpressed KIAA1363 or CES1 proteins were
diluted to a final protein concentration of 0.1 mg/ml per well. In
parallel, equivalent amounts of mock-transfected cell lysate protein
were also assayed in order to subtract the intrinsic 4-MUBA hy-
drolytic activity of the COS-7 lysate/buffer mix.
Working solutions of the oxons were diluted in ethanol and
added to the reaction mixture to give the desired concentrations.
The final volume of ethanol in the wells was 1.5% (v/v); this amount
of ethanol did not affect enzymatic activity. KIAA1363 and CES1
enzymatic reaction rates were corrected by subtracting the mock-
transfected COS-7 lysate hydrolysis rate. For IC50 measurements,
the enzyme and oxon were incubated for either 0 min or 15 min at
37 ꢂC, followed by addition of 4-MUBA (final concentration

74
M.K. Ross et al. / Archives of Biochemistry and Biophysics 590 (2016) 72e81
250
m
M). Reaction progress was monitored by measuring the
effectively reduces the affinity of oxon for the enzyme (Fig. 5A);
therefore, Ki is the corrected dissociation constant for E$I and is
calculated as follows.
fluorescence of the hydrolysis product 4-methyumbelliferone (ex.
302 nm, em. 355 nm) for 5 min at 37 ꢂC. The initial slopes of the
progress curves were determined and used to calculate enzymatic
activities. The curves were linear during the 5 min reaction period.
IC50 values were determined by plotting the % inhibition of enzyme
activity versus the oxon concentration. % inhibition was defined as:
(rate of reaction with no oxon e rate of reaction with oxon)/rate of
reaction with no oxon ꢀ 100. IC50 values were interpolated from the
curves.
À
Áꢂ
ki ¼ Ki0 ½ð1 þ ½Sꢃ=KmÞꢃ
(4)
Ki0 is the apparent dissociation constant for E$I, [S] is the 4-
MUBA concentration (250
mM), and Km is the Michaelis constant
for 4-MUBA (227 M, determined in the absence of oxon). Thus, the
m
corrected bimolecular rate constant can be expressed as: kinact
[Ki ꢀ (1 þ [S]/Km)].
/
In the linear steady-state phase of the reaction of 4-MUBA hy-
drolysis, the rates of enzyme phosphorylation and dephosphory-
lation are equal [22]. Thus, the term vss is a measure of the steady-
state turnover of oxon and a function of [I], as shown in Eqn. (5).
2.4. Kinetic studies
A competitive kinetic scheme describing the covalent inhibition
of serine hydrolases (E) by oxons (I) (and its reactivation) in the
presence of ester substrate (S) is shown in Fig. 5A. To determine the
bimolecular rate constants for enzyme inactivation, the oxon (at
ꢀ
ꢁ ꢀ
ꢁ
vss
¼
v0 ꢀ KmI þ vmin ꢀ ½Iꢃ = KmI þ ½Iꢃ
(5)
various concentrations) and 4-MUBA (250 mM) were added to re-
v0 is the phosphorylation rate extrapolated to zero inhibitor con-
centration, vmin is the asymptotic rate at infinite inhibitor concen-
tration, and KmI; is the Michaelis constant for KIAA1363-catalyzed
hydrolysis of the OP oxon [24]. Values for v0, vmin, and KmI were
obtained from least-squares fits of the data to Eqn. (5).
The reactivation (dephosphorylation) rate constant (kreact) was
calculated using Eqn. (6), which can be rearranged to give Eqn. (7)
[25], where VS is the velocity of the reporter substrate (4-MUBA,
action buffer and prewarmed at 37 ꢂC (5 min). Cell lysate containing
overexpressed KIAA1363 or CES1 protein (0.1 mg/ml final concen-
tration) was added to initiate the reaction. Progress of the reaction
was monitored for 45 min.
For inhibition of CES1, the reaction curves were fit to Eqn. (1),
which assumes that no reactivation of enzyme occurs during the
45 min time period. This assumption is supported by the half-life of
reactivation of pure CES1-diethylphosphate protein (t1/2 ¼ 31 h) [7].
250
mM) hydrolysis reaction in the absence of inhibitor. It was
ꢀ
ꢁ
Ft ¼ F0 þ ðF∞ ꢁ F0Þ ꢀ 1 ꢁ eꢁkobsꢀt
(1)
assumed that the rate of the “aging” reaction was insignificant (see
Discussion for more explanation).
Data were fitted using SigmaPlot 8.0, and the value for the
apparent first-order rate constant of enzyme inactivation (kobs) was
determined at each oxon concentration. F0 is fluorescence at t ¼ 0,
Ft is fluorescence at time t, F∞ is fluorescence at time infinity, t is
vmin=VS ¼ kreact=ðkinact þ kreact
Þ
(6)
(7)
kreact ¼ kinact ꢀ vmin=ðVS ꢁ vminÞ
time in s, and kobs is the observed rate constant in sꢁ1
.
Evidence exists that KIAA1363 can completely turnover 1 nM
CPO in 60 min (100% hydrolysis) [9]. Thus, we fitted the kinetic data
for KIAA1363 to a model that includes enzyme reactivation [22], as
given in Eqn. (2).
2.5. Inhibition of KIAA1363 or CES1 determined using the activity-
based protein probe FP-biotin
ꢀ
ꢁ
obsꢀt
Ft ¼ F0 þ ½ðv0 ꢁ vssÞ=kobsꢃ ꢀ 1 ꢁ eꢁk
þ vss ꢀ t
(2)
KIAA- or CES1-transfected COS7 cell lysates (1 mg/ml protein,
25
for 15 min with varying concentrations of CPO, then treated with
FP-biotin (2 M final; 1
l of a 25 ꢀ stock dissolved in DMSO) for 1 h
at room temperature. Reactions were quenched by adding 10 L of
ml reaction volume, 50 mM TriseHCl, pH 7.4) were preincubated
F0 is fluorescence at t ¼ 0, kobs is the observed first-order inhi-
bition rate constant, v0 is the initial velocity of the 4-MUBA hy-
drolysis reaction, and vss is the steady-state velocity. The non-linear
pre-steady state phase of the reaction depicts the progressive in-
hibition of enzyme by oxon, which is represented by the time-
dependent decrease in the slope of the progress curve. In the
linear steady-state phase of the reaction, during which the reaction
velocity equals vss, the rates of enzyme phosphorylation and
dephosphorylation are considered to be equal [22].
m
m
m
6 ꢀ SDS-PAGE loading buffer (reducing) and heating the samples
for 5 min (95 ꢂC). Biotinylated proteins were resolved by SDS-PAGE
(10%), transferred to a PVDF membrane, and detected with avidin-
HRP as described in detail in [26]. Densitometry of bands was used
to estimate IC50 values.
2.6. Reactivation of oxon-inhibited KIAA1363 activity
The kobs values obtained were then plotted against the oxon
concentration and fitted to Eqn. (3) [23].
KIAA-transfected COS7 cell lysate (1 mg/ml protein, 50
tion volume, 50 mM TriseHCl, pH 7.4) was pre-incubated with CPO
(1 M final concentration; ethanol vehicle, 2% v/v) for 15 min at
ml reac-
À
Á
kobs ¼ ðkinactÞ ꢀ ½Iꢃ= Ki0 þ ½Iꢃ
(3)
m
kinact is the first-order rate constant for the inactivation (phos-
phorylation) of the enzyme by the oxon, Ki0 is the apparent disso-
ciation constant for the reversible E$I (enzyme-oxon) complex, and
[I] is the inhibitor (oxon) concentration. Because KIAA1363 and
CES1 were not purified from transfected COS-7 cell lysates, the true
bimolecular rate constant is not known and therefore the apparent
bimolecular rate constant is kinact/Ki0. However, the apparent
dissociation constant (Ki0) between enzyme and oxon can be cor-
rected by Eqn. (4), which takes the reporter substrate 4-MUBA into
account. The presence of 4-MUBA in the enzymeeoxon reaction
37 ꢂC. A parallel control reaction containing KIAA-transfected COS7
cell lysate was treated with ethanol (2% v/v). The reactions were
then diluted 1:100 (v/v) with 50 mM TriseHCl (pH 7.4) buffer and
incubated for an additional 60 min. Aliquots were removed every
10 min to assay the residual 4-MUBA hydrolysis activity.
2.7. Statistical analysis
SigmaStat was used to perform Student's t-test and linear
regression analysis.

M.K. Ross et al. / Archives of Biochemistry and Biophysics 590 (2016) 72e81
75
3. Results
15 min prior to addition of 4-MUBA. The oxons potently inhibited
KIAA1363 activity with IC50 values in the low nanomolar range
(Fig. 3). On the basis of IC50s, CPO was a more potent inhibitor than
PO of KIAA1363 and CES1. The IC50 values obtained following a
15 min incubation of enzyme and inhibitor were lower than IC50
values obtained following the 0 min incubation (Fig. 3), which is
consistent with a time-dependent covalent reaction between the
catalytic serine residue of KIAA1363 and oxon. Moreover, CES1 was
slightly more sensitive than KIAA1363 to the inhibitory effects of
both oxons. In contrast to CPO and PO, MPO was a relatively poor
inhibitor of KIAA1363 (IC50 > 100 nM) and was not further studied.
In the second approach, KIAA1363- or CES1-transfected cell ly-
sates were treated with CPO for 15 min, followed by addition of the
activity probe FP-biotin (Fig. 4). Using the competitive activity-
based protein profiling (ABPP) assay, somewhat higher IC50 values
were obtained compared with the spectrofluorometric method
(Fig. 3). Nevertheless, both approaches indicated that KIAA1363
was potently inhibited by nanomolar concentrations of CPO.
3.1. High-throughput biochemical assay of KIAA1363 activity
Overexpression of KIAA1363 protein in COS-7 cells was verified
by serine hydrolase activity profiling (Fig.1A,B) and enzyme activity
assays (Fig. 1C). Following reaction with FP-biotin, KIAA1363-
transfected COS-7 cell lysate exhibited a doublet band on the blot
(~45e50 kDa), which was absent when the lysate was denatured
prior to adding FP-biotin (Fig. 1A). The ester-containing reporter
substrates 4-MUBA and pNPV were hydrolyzed by KIAA1363; 4-
MUBA was cleaved ~5 ꢀ faster than pNPV (Fig. 1C). Because the
putative physiological substrates for KIAA1363 (AcMAGE and cho-
lesteryl esters) do not lend themselves to a high-throughput assay
format, we designed a 96-well spectrofluorometric plate assay
using the substrate 4-MUBA to evaluate the potency of inhibition of
KIAA1363 by OP oxons, as measured by IC50s (Fig. 2).
3.2. Inhibitor potency: IC50 determination
3.3. Corrected bimolecular rate constants (kinact/Ki) of KIAA1363
inhibition by CPO and PO
IC50s for CPO and PO toward KIAA1363 were compared with
those for CES1, a related serine hydrolase that was also expressed in
COS-7 cells. Two approaches were used to estimate the IC50 values
of the oxons. First, the spectrofluorometric method was used to
evaluate the inhibition of either KIAA1363- or CES1-transfected cell
lysates by the oxons following either a 0 or 15 min pre-incubation
period prior to the addition of substrate. The 0 min pre-incubation
indicates that the oxon and ester substrate (4-MUBA) see the
enzyme at the same time (i.e., simultaneously), whereas the 15 min
pre-incubation indicates that the enzyme is treated with oxon for
The corrected bimolecular rate constants were determined for
the oxons and KIAA1363. Using the approach outlined by Crow et al.
[7], the bimolecular rate constant for either CPO or PO and
KIAA1363 was determined by titrating KIAA1363-transfected COS-
7 cell lysate with oxon in the presence of 4-MUBA. The fluorescence
of 4-methylumbelliferone produced by the hydrolysis of 4-MUBA in
mock-transfected COS-7 cell lysates was determined in parallel on
the same multi-well plate and subtracted from the KIAA1363
Fig. 1. Expression of recombinant human KIAA1363 in COS7 cells. (A) COS7 cells were mock-transfected or transfected with plasmid containing human KIAA1363 cDNA. After cell
lysis, serine hydrolases were labeled with the activity probe FP-biotin (2 M, 1 h, room temperature), followed by SDS-PAGE and avidin-HRP blotting to detect biotinylated proteins.
m
KIAA1363 doublet bands are indicated. * indicates an endogenous biotinylated protein in COS7 cell lysates. Negative control reactions included cell lysates that were preheated (pre-
heat) before addition of FP-biotin. (B) Concentration dependence of the FP-biotin reaction with KIAA1363-transfected COS7 cell lysates. The indicated concentration of FP-biotin was
incubated with cell lysate for 1 h at room temperature. * indicates an endogenous biotinylated protein in COS7 cell lysates. (C) Hydrolysis activity of KIAA1363- (KIAA1363) and
mock-transfected (control) COS7 cell lysates using the substrates pNPV and 4-MUBA. Data represent mean standard deviation (n ¼ 3). *, p < 0.05, KIAA1363 versus control,
Student's t-test.

76
M.K. Ross et al. / Archives of Biochemistry and Biophysics 590 (2016) 72e81
Fig. 2. Format to measure KIAA1363 or CES1 activities using a high-throughput fluorescence assay. (A) Inhibitor (oxon, variable concentrations) and substrate (4-MUBA, 250 mM
final concentration) were added to each well of a 96-well plate and pre-warmed for 5 min (37 ꢂC), followed by addition of the serine hydrolase (KIAA1363- or CES1-transfected or
mock transfected cell lysate) to initiate the hydrolytic reaction. (B) Progress of the hydrolysis reaction catalyzed by recombinant serine hydrolase was monitored by fluorescence (F).
(C) Each progress curve represents the corrected F, as indicated by the red solid curve (no oxon) and red dashed curve (1 nM oxon), and was monitored for 45 min. Values from
triplicate wells were averaged and the mock-lysate wells were subtracted from the hydrolase-lysate wells. The rate of reaction between oxon and enzyme is slowed by the presence
of 4-MUBA, thereby enabling kobs (i.e., first-order rates of enzyme inactivation) to be obtained by fitting the progress curves to Eqs. 1 or 2 (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article).
Fig. 3. Potency of inhibition of CES1 and KIAA1363 by CPO and PO determined by fluorescence assay. IC50 values were determined with (þ) or without (ꢁ) a pre-incubation
period with oxon (37 ꢂC, 15 min) prior to the addition of substrate (4-MUBA). Data represent the means standard deviation of 2e3 independent experiments. Data are presented in
both graphical (left) and tabular (right) forms.
lysate-catalyzed reactions (Fig. 2A).
determined at each CPO concentration were corrected by sub-
tracting the apparent kobs determined in the absence of inhibitor,
which represents the progressive inactivation of enzyme activity in
the absence of oxon (possibly due to slight denaturation of
KIAA1363 over the 45 min duration of the assay). This correction
yields the kobs (corrected) values.
Values for kobs (corrected) were plotted against the appropriate
concentration of oxon and fit to Eqn. (3) to obtain kinact and Ki
(Fig. 6A), and the corrected bimolecular rate constants (kinact/Ki) for
The kinetic scheme describing the inhibition of a serine hy-
drolase by oxon, which can undergo reactivation, in the presence of
an ester-containing reporter substrate is shown in Fig. 5A. Progress
curves for KIAA1363-catalyzed 4-MUBA hydrolysis in the presence
of increasing concentrations of CPO are shown in Fig. 5B. Each
progress curve was fit to an equation that describes the inactivation
and spontaneous reactivation of the enzyme (Eqn. (2)) to obtain the
apparent first-order rate constant of inactivation (kobs). kobs values

M.K. Ross et al. / Archives of Biochemistry and Biophysics 590 (2016) 72e81
77
Fig. 4. Potency of inhibition of KIAA1363 and CES1 by CPO determined using the serine hydrolase activity-based probe, FP-biotin. (A) Scheme describing the strategy to
characterize CPO-mediated inactivation of KIAA1363 (and CES1) by activity-based protein profiling (ABPP). The activity probe, FP-biotin, is shown in simplified form with a reactive
warhead (black circle) attached to a biotin (B) tag (green star). Recombinant CES1 (B) and KIAA1363 (C) proteins were incubated with the indicated concentrations of CPO for 15 min
(37 ꢂC), followed by addition of FP-biotin (2
mM, 1 h, room temperature). After separation of proteins by SDS-PAGE and avidin-HRP blotting, the intensity of the biotin-labeled CES1
and KIAA1363 proteins were quantified and plotted versus CPO concentration. IC50 values were interpolated from the curves and are indicated on the graphs. Data are repre-
sentative of two independent experiments (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 5. Effect of the substrate on the progressive inhibition of KIAA1363 by oxon. (A) General kinetic scheme that describes the inhibition and reactivation of serine hydrolases (E) by
oxons (I) in the presence of an ester substrate (S). The turnover number kcat for the ester substrate (S) is a function of the rates of acylation (k2) and deacylation (k3) [i.e.,
kcat ¼ k2 ꢀ k3/(k2 þ k3)] [24]; however, these steps are not explicitly shown for simplicity. (B) Inhibition of KIAA1363 by varying concentrations of CPO. The progress of the 4-MUBA
hydrolysis reaction was followed by measuring the fluorescence of 4-methylumbelliferone produced during the 45 min monitoring period. Inset, representative experiment
describing the inhibition of KIAA1363-catalyzed 4-MUBA hydrolysis by CPO. KIAA1363-transfected cell lysate was added to a prewarmed mixture of CPO and 4-MUBA (i.e., pre-
incubation time was 0 min between CPO and KIAA1363 prior to the addition of substrate). Reaction progress was monitored by fluorescence for 5 min at 37 ꢂC.
the reaction of KIAA1363 with either CPO or PO could be calculated.
The kinetic values are reported in Table 1. On the other hand, data
obtained with CES1 and oxon inhibitors did not fit a hyperbolic
equation; instead, a linear equation was used to fit the data (Fig. 6B;
data for CPO shown). The bimolecular rate constants for the oxons
and CES1 are also reported in Table 1. Although the kinact/Ki for CPO/

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M.K. Ross et al. / Archives of Biochemistry and Biophysics 590 (2016) 72e81
Fig. 6. Concentration-dependent inactivation of either KIAA1363 or CES1 by CPO. The observed first-order inhibition rate constant (kobs) was plotted against CPO concentration.
For KIAA1363 (A), data were fitted with Eqn. (3): kinact ¼ 0.0062 sꢁ1; Ki ¼ 0.76 nM. For CES1 (B), data were fitted with Eqn. (3), assuming that Ki (CPO) >> [I]: kinact
/
Ki ¼ 2.5 ꢀ 106 sꢁ1 Mꢁ1. The graphs are representative of the type of data used to determine the kinetic parameters.
Table 1
Corrected kinetic constants for reactions of KIAA1363 and CES1 with CPO or PO.
Kinetic parameters a
Enzyme
Oxon
kinact (sꢁ1
)
kic (M)
kinact/Ki (sꢁ1 Mꢁ1
)
kreact (sꢁ1
)
KIAA1363
CES1
KIAA1363
CES1
CPO
CPO
PO
0.0069 0.0007
d b
1.6 ꢀ 10ꢁ9
4.4( 0.4) ꢀ 106
6.7( 4.2) ꢀ 106
0.4 ꢀ 106
0.00043 0.00019
d b
d b
0.00092
d b
2.9 ꢀ 10ꢁ9
d b
0.0003
d b
PO
1.5 ꢀ 106
a
Values are means SD of 2e4 independent experiments, each with triplicate reactions for each oxon concentration.
Not determined because curves were linear, not hyperbolic (see Fig. 6B for CPO data).
Corrected ki ¼ ðKi0Þ=½ð1 þ ½Sꢃ=KmÞꢃ. [S] ¼ 250
b
c
m
M.Km4ꢁMUBA ¼ 227mm.
CES1 overexpressed in COS7 cells (6.7 ꢀ 106 sꢁ1
M
ꢁ1) is slightly
bimolecular rate constant for the reaction between PO and
KIAA1363 (Table 1). The kinact for PO was 7.5-fold lower than kinact
for CPO (Table 1), indicating CPO is more potent than PO at inhib-
iting KIAA1363. In addition, by analyzing the data using Eqns. (5)
and (6), the ratio kinact/kreact for the reaction between PO and
KIAA1363 was calculated to be 3.4, whereas (as mentioned above)
this ratio was 16 for the reaction of CPO with KIAA1363. Using the
empirical kinact value for the PO and KIAA1363 reaction (Table 1),
the kreact for the reaction was calculated to be 0.0003 sꢁ1
(kreact ¼ kinact/3.4). Because both CPO and PO produce the same
modification on the active-site serine residue (diethylphosphor-
ylation) in KIAA1363, then the “true” kreact must be identical in each
case. The calculated kreact values obtained for CPO-KIAA1363
lower than that previously reported for CPO and purified CES1 [7],
this is not surprising given that our current data was obtained using
CES1 protein in a crude cell lysate. We used CES1 in a crude lysate to
compare results directly with KIAA1363, because it was also in a
crude lysate (i.e., not purified).
In addition to determining kinact/Ki, fitting the progress curves
for CPO-inhibited KIAA1363 activity to Eqn. (2) permitted estimates
of the steady-state velocity (vss) to be obtained. The various values
of vss were then plotted against the corresponding CPO concen-
tration (I) and the data fit to Eqn. (5) to obtain estimates for v0, vmin
,
and KmI , as described in the Methods section. The reactivation rate
constant, kreact, could then be calculated using Eqn. (7). This value is
also reported in Table 1. The ratio kinact/kreact (phosphorylation/
dephosphorylation) for CPO and KIAA1363 was 16, which indicated
that the rate of inactivation of KIAA1363 by CPO was 16 ꢀ faster
than the rate of reactivation.
(0.0004 sꢁ1
) ) are reasonably
and PO-KIAA1363 (0.0003 sꢁ1
similar to one another, which is consistent with this premise.
Attempts to directly measure the rate of KIAA1363 reactivation
(kreact), as was previously done for CES1 by treating the KIAA1363
with excess oxon followed by removal of the unreacted oxon using
a desalting spin filter [7], were stymied by the fact that control
KIAA1363 activity was lost during the desalting process suggesting
either enzyme instability or adsorption to the spin filter. Therefore,
an alternative approach was used: KIAA1363 was treated with
As shown in Fig. 7, differences in the shapes of the inhibition
progress curves for KIAA1363 and CES1 in the presence of a com-
mon oxon (paraoxon) were apparent. It is known that CES1 is
essentially irreversibly inactivated by paraoxon in the time frame
considered here [7], which is exemplified by the time-dependent
decrease in the slopes (deceleration) for each curve seen in
Fig. 7A. In contrast to CES1, the slope of the reaction curves for
KIAA1363 do not approach zero but instead have a more linear
appearance with a positive slope (Fig. 7B), even at high PO con-
centrations (100 nM), suggesting a steady-state turnover of the
oxon by KIA1363. Thus, a constant (steady state) amount of free
enzyme remains available to catalyze the hydrolysis of 4-MUBA
even at high PO concentrations, unlike what is seen for CES1
which is inactivated. Nevertheless, we estimated the corrected
excess CPO (1 mM) or vehicle (control) for 15 min, then the reaction
mixtures were immediately diluted 100-fold and incubated for up
to 60 min (at 37 ꢂC). Aliquots were removed every 10 min and
KIAA1363 activity determined using the substrate 4-MUBA. The
kreact value determined by this approach was 0.00038 sꢁ1 (Fig. 8).
This result supported the notion that KIAA1363 can be reactivated
(t1/2 of reactivation ¼ 30.4 min) following its inhibition by oxons. It
also validated the calculated kreact in Table 1. It should be noted,
however, that this approach does not remove unreacted oxon;

M.K. Ross et al. / Archives of Biochemistry and Biophysics 590 (2016) 72e81
79
Fig. 8. Reactivation of CPO-inhibited KIAA1363. KIAA-transfected COS7 cell lysate
(1 mg/ml protein) was pre-incubated with CPO (1 M final concentration) for 15 min at
m
37 ꢂC. A parallel control reaction containing KIAA-transfected COS7 cell lysate was
treated with ethanol vehicle. Reactions were then diluted 1:100 (v/v) with 50 mM
TriseHCl (pH 7.4) and aliquots removed at the indicated times to assay the residual 4-
MUBA hydrolysis activity for a total incubation time of 60 min.
low level non-KIAA1363-derived signals that can be subtracted
from the KIAA1363-transfected cell lysate. As shown in Fig. 2, the
intrinsic hydrolysis activity in the control (“background”) cell ly-
sates was subtracted from the hydrolysis activity in the KIAA1363-
transfected cell lysates. Thus, the corrected hydrolysis activity we
measured using the substrate 4-MUBA can be attributed to the
activity of KIAA1363. It should be noted that other studies of
esterase inhibition by organophosphorus compounds have been
done in crude tissue lysates. For example, Estevez et al. used the
cytosolic (soluble) fraction from either chicken brain or sciatic
nerve to examine the inhibition of neuropathy target esterase (NTE)
by paraoxon [27]. Ki and IC50 values were derived using this crude
mixture of enzymes. In addition, Quistad et al. used a variety of
sources, including bovine pancreas, rat serum, and bovine milk, to
study the inhibition of several esterases by OP poisons [28]. Thus,
our approach using an enzyme that was overexpressed in cell ly-
sates was appropriately controlled by the use of control cell lysates
and has precedence in the literature.
Pharmacokinetic modeling of OP insecticides requires kinetic
characterization of the interaction of the OP with enzymes
responsible for its metabolism. Here, we determined the potencies
and rate constants of CPO and PO with respect to their ability to
inhibit KIAA1363, which is one of the most abundant serine hy-
drolases found in mouse brain and an important detoxication
enzyme of OPs [10]. We found that KIAA1363 was highly sensitive
to inhibition by the oxons, exhibiting IC50s between 1 and 17 nM,
depending on the experimental approach used. These data are in
line with IC50s previously reported for KIAA1363 and oxons when
using mouse brain membranes and 1 nM [3H]-CPO to radiolabel
serine hydrolases [10]. In addition, we determined the corrected
bimolecular rate constants for the reactions of the oxons with both
KIAA1363 and CES1, which is another detoxification enzyme. The
corrected kinact/Ki for CPO and KIAA1363 was ~10-fold higher than
for PO and KIAA1363, whereas the bimolecular rate constants for
the reaction of CPO with either KIAA1363 or CES1 were similar to
each other (Table 1). These results indicated that KIAA1363 and
CES1 are both equally sensitive to CPO. Although kinetic
Fig. 7. Effect of the substrate on the progressive inhibition of CES1 and KIAA1363
by oxon. Inhibition of CES1- (A) and KIAA1363-catalyzed (B) hydrolytic activity by
varying concentrations of PO. The progress of the 4-MUBA hydrolysis reaction was
followed by measuring fluorescence for 45 min.
although the oxon is diluted by a factor of 100, it is possible that it
will re-inactivate KIAA1363. Further, the substantial dilution of
enzyme also causes a technical issue to arise with regard to the
ability to measure its activity (i.e., the signal-to-noise ratio of the
spectrofluorometric method is reduced). These are two technical
limitations of this method and why more emphasis should be given
to the calculated kreact values in Table 1.
4. Discussion
A possible limitation of obtaining kinetic parameters for a spe-
cific reaction in cell lysates is that the measured values, i.e. Vmax for
a particular enzyme, will be the sum of activities for all isoenzymes
of that particular enzyme. This is a major issue for drug metabo-
lizing enzymes such as cytochrome P450s (CYPs) that have multiple
isozymes that catalyze the conversion of a specific substrate to a
particular product. Therefore, using isolated enzymes for the CYPs
is important for determining the intrinsic kinetic parameters for
each isoform. However, in the case of KIAA1363, there are no other
known isozymes for this enzyme. In addition, on the basis of the
gel-based ABPP profiling (Fig. 1), we have verified that the native
COS7 cells (the recipient cells of the KIAA1363-containing plasmid)
do not express KIAA1363. Thus, our “background” cell lysate yields

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M.K. Ross et al. / Archives of Biochemistry and Biophysics 590 (2016) 72e81
parameters for the interaction of CPO with CES1 have been re-
ported [7], they have not been for CPO and KIAA1363. Assuming
that kcat z kreact for the catalytic turnover of CPO by KIAA1363, i.e.,
the rate limiting step is the reactivation of the inhibited enzyme,
which is a reasonable assumption for esterase-oxons because
typically kreact << kinact for this type of reaction, and KmCPO ¼ 4:7nm
(which was obtained by fitting data to Eqn. (5)), the apparent cat-
alytic efficiency (kcat/Km) of CPO turnover by KIAA1363 was calcu-
conclusions can be made for PO and KIAA1363. Therefore,
KIAA1363 is very efficient at detoxifying oxons, and is likely an
important enzyme that protects against OP poisoning in humans,
particularly in the brain because of its high expression. Further-
more, our data indicated that the KIAA1363-diethylphosphate
protein is reactivated much faster than the CES1-
diethylphosphate protein (t1/2 ¼ 0.5 h for KIAA1363 versus t1/
¼ 31 h for CES1 [8]). Thus, although carboxylesterases can reac-
2
lated to be 9.1 ꢀ 104 Mꢁ1
s
ꢁ1. Interestingly, the catalytic efficiency
tivate following inactivation by select OPs [34,35], KIAA1363 ap-
pears to be a more efficient OP hydrolase.
for CPO hydrolysis by KIAA1363 is similar to human paraoxonase
(PON1), an important enzyme that detoxifies CPO [29]. For
example, catalytic efficiencies for the hydrolysis of CPO by recom-
binant PON1Q192 and PON1R192 isoforms were 9 ꢀ 104 Mꢁ1 sꢁ1 and
Although liver is a notable exception, KIAA1363 is widely
distributed in several tissues including the brain [9]. The develop-
mental expression (or ontogeny) of KIAA1363 in tissues is un-
known. Whether changes in sensitivity to OP insecticides during
development correlates with the expression levels of KIAA1363 is
an unanswered but intriguing question. To our knowledge PBPK/PD
models of OP metabolism in rodents and humans do not incorpo-
rate the metabolism of OPs by KIAA1363, which could be an
important pathway of detoxication in tissues such as the brain.
Given the high sensitivity of KIAA1363 to oxons, incorporating this
pathway into PBPK/PD models that evaluate the toxicokinetics of
oxons seems warranted. Thus, characterizing the interactions be-
tween oxons and the enzymes, such as KIAA1363, that detoxify
these bioactive molecules is a useful addition to the knowledge-
base. The kinetic parameters that were determined here should
help the development of more sophisticated PBPK models, which
take into account detoxication reactions occurring in sensitive
target tissues, such as brain. The bimolecular rate constants and
IC50s that we determined for CPO and PO with human KIAA1363
should prove useful in these modeling efforts.
15 ꢀ 104 Mꢁ1
s
ꢁ1, respectively [30]. Whereas PON1 is abundantly
expressed in liver and blood, KIAA1363 is not [9], thus KIAA1363
will not impact the pharmacokinetics of oxons to the extent that
PON1 does [30]. Therefore, it can be concluded that PON1 (and
probably CES1 due to its abundant levels in liver) is a more
important factor in overall CPO metabolism in vivo than KIAA1363.
Nevertheless, on the basis of its robust expression in brain and
sensitivity to oxons, it is likely that KIAA1363 has an important
protective function in this organ [10]. Our results lend further
support to this notion.
Previous work [10] indicated that maximal radiolabeling of
KIAA1363 in mouse brain membranes by [3H]-CPO (1 nM) occurred
at 5 min; 48 mol % of the added CPO was covalently bound to
KIAA1363 [4 mol % was present as the monoethylphosphoryl
adduct (“aged” adduct), while 44 mol % was present as the dieth-
ylphosphoryl adduct (non-aged adduct)]. By 60 min, the amount of
covalent phosphoryl adduct on KIAA1363 was reduced to 25 mol %
of the added CPO [12 mol
% was present as the mono-
ethylphosphoryl adduct, while 13 mol % was present as the dieth-
ylphosphoryl adduct]. The remaining CPO (75 mol %) had been
completely hydrolyzed by KIAA1363 to trichloropyridinol and
diethylphosphate products. Because “aging” of the dieth-
ylphosphoryl adduct accounted for only a small fraction (12 mol %)
of the overall metabolism of 1 nM CPO by KIAA1363 during the
60 min incubation period, this reaction was not incorporated into
our kinetic analysis. This assumption considerably simplified the
equations needed to model covalent inhibition and spontaneous
reactivation of KIAA1363, as has been discussed previously for es-
terases in general [31]. Furthermore, it should be noted that in
another study [9], “aging” of the CPO-modified KIAA1363 was not
apparent, because CPO was hydrolyzed completely by KIAA1363
during the 60 min incubation. This provided further justification for
not including the aging step in the kinetic equations.
KIAA1363 is an integral membrane protein in the endoplasmic
reticulum [18]. It appears to be a lipase with acetyl mono-
acylglycerol ethers (AcMAGEs) and cholesteryl esters as putative
substrates [12,32]. Inhibition of KIAA1363 in macrophages has been
shown to lower production of pro-inflammatory cytokines through
changes in key ether lipid metabolism pathways [33]. It should also
be noted that other studies did not observe a cholesteryl esterase
activity associated with KIAA1363 [14]. We also tested the ability of
KIAA1363 to hydrolyze both cholesteryl oleate and 4-
methylumbelliferyl oleate, but could not detect any activity with
either substrate (data not shown). More work is required to identify
the endogenous substrates of KIAA1363.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgments
Research support was provided by NIH 1R15ES015348-01A1, NIH
1R15ES015348-02, 3R15ES015348-01A1S1, and 3R15ES015348-
01A1S2. Kim Pluta was supported by NIH T35RR007071 (Summer
Research Experience program for veterinary students). We also
gratefully acknowledge the assistance of Shuqi Xie.
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