Liver prenylated methylated protein methyl esterase is the same enzyme as Sus scrofa carboxylesterase

Article abstract of DOI:10.1002/jbt.20214

The C-terminal -COOH of prenylated proteins is methylated to -COOCH ₃. The -COOCH₃ ester forms are hydrolyzed by prenylated methylated protein methyl esterase (PMPMEase) to the original acid forms. This is the only reversible step of the prenylation pathway. PMPMEase has not been purified and identified and is therefore understudied. Using a prenylated-L-cysteine methyl ester as substrate, PMPMEase was purified to apparent homogeneity from porcine liver supernatant. SDS-PAGE analysis revealed an apparent mass of 57 kDa. Proteomics analyses identified 17 peptides (242 amino acids). A Mascot database search revealed these as portions of the Sus scrofa carboxylesterase, a 62-kDa serine hydrolase with the C-terminal HAEL endoplasmic reticulum-retention signal. It is at least 71% identical to such mammalian carboxylesterases as human carboxylesterase 1 with affinities toward hydrophobic substrates and known to activate prodrugs, metabolize active drugs, as well as detoxify various substances such as cocaine and food-derived esters. The purified enzyme hydrolyzed benzoyl-Gly-farnesyl-L-cysteine methyl ester and hydrocinamoyl farnesyl-L-cysteine methyl ester with Michaelis-Menten constant (K_m) values of 33 ± 4 and 25 ± 4 μM and V _max values of 4.51 ± 0.28 and 6.80 ± 0.51 nmol/min/mg of protein, respectively. It was inhibited by organophosphates, chloromethyl ketones, ebelactone A and B, and phenylmethylsulfonyl fluoride.

Full text of DOI:10.1002/jbt.20214

J BIOCHEM MOLECULAR TOXICOLOGY
Volume 22, Number 1, 2008
Liver Prenylated Methylated Protein Methyl Esterase Is
the Same Enzyme as Sus scrofa Carboxylesterase
Onovughode T. Oboh and Nazarius S. Lamango
College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA; E-mail: nazarius.lamango@famu.edu.
Received 25 July 2007; revised 28 August 2007; accepted 1 September 2007
ABSTRACT: The C-terminal COOH of prenylated
proteins is methylated to COOCH3. The COOCH3
ester forms are hydrolyzed by prenylated methylated
protein methyl esterase (PMPMEase) to the original
acid forms. This is the only reversible step of the
prenylation pathway. PMPMEase has not been pu-
rified and identified and is therefore understudied.
Using a prenylated-L-cysteine methyl ester as sub-
strate, PMPMEase was purified to apparent homogene-
ity from porcine liver supernatant. SDS-PAGE analy-
sis revealed an apparent mass of 57 kDa. Proteomics
analyses identified 17 peptides (242 amino acids). A
Mascot database search revealed these as portions of
the Sus scrofa carboxylesterase, a 62-kDa serine hydro-
lase with the C-terminal HAEL endoplasmic reticulum-
retention signal. It is at least 71% identical to such mam-
malian carboxylesterases as human carboxylesterase
1 with affinities toward hydrophobic substrates and
known to activate prodrugs, metabolize active drugs,
as well as detoxify various substances such as co-
caine and food-derived esters. The purified enzyme hy-
drolyzed benzoyl-Gly-farnesyl-L-cysteine methyl es-
ter and hydrocinamoyl farnesyl-L-cysteine methyl es-
ter with Michaelis–Menten constant (Km) values of
33 4 and 25 4 µM and Vmax values of 4.51 0.28 and
6.80 0.51 nmol/min/mg of protein, respectively. It was
inhibited by organophosphates, chloromethyl ketones,
ebelactone A and B, and phenylmethylsulfonyl fluo-
KEYWORDS: Carboxymethyl Esterase; Prenylation;
Organophosphates; Prenylated Proteins; Hydrolase;
Ebelactone
INTRODUCTION
The estimated 2% of eukaryotic proteins that
are prenylated [1] undergo S-adenosyl-L-methionine
(SAM)-dependent methylation to form carboxylmethyl
esters in a reaction catalyzed by prenylated protein
methyl transferase (PPMTase). This reaction is the only
reversible step in the prenylation pathway as the preny-
lated protein carboxylmethyl esters are then the sub-
strates of the prenylation-dependent esterase, preny-
lated methylated protein methyl esterase (PMPMEase),
which hydrolyzes the ester to form the original un-
methylated proteins [2,3]. These reactions are signifi-
cant with respect to the conformations of the prenylated
proteins since the reversibility of the process entails
the constant removal and reintroduction of a negative
charge in the close vicinity of the isoprenyl moiety.
The dynamic equilibrium may be critical to the bio-
chemical activity of the proteins since the interaction
of the prenylated proteins with other macromolecules
may be significantly affected by the methylation state
of the adjacent –COOH group. This is perhaps more
substantial based on the fact that the isoprenyl group
of proteins such as the G-protein γ subunits are es-
sential for their coupling of receptor-derived signals
to such effector enzymes as the adenylyl cyclases and
phospholipase Cβ2 [2,4,5]. Furthermore, an isoprenyl-
binding site has been identified using solution NMR on
Rho dissociation inhibitor [6]. Prenyl-L-cysteine (PC)
analogs that mimic only the C-terminal prenylated
cysteine residue of prenylated proteins exert physio-
logical effects such as the inhibition of platelet aggre-
gation [2] or SAM-induced Parkinson’s disease-like ef-
fectsinratsthroughmechanismsthat donot involve the
ride. ꢀ 2008 Wiley Periodicals, Inc. J Biochem Mol Toxi-
C
col 22:51–62, 2008; Published online in Wiley InterScience
(www.interscience.wiley.com). DOI 10:1002/jbt.20214
Correspondence to: Nazarius S. Lamango.
Contract Grant Sponsor: NIH/NIGMS/MBRS/SCORE.
Contract Grant Number: GM 08111-35.
Contract Grant Sponsor: Pharmaceutical Research Center
NIH/NCRR.
Contract Grant Number: G12 RR0 3020.
2008 Wiley Periodicals, Inc.
c
ꢀ
51

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OBOH AND LAMANGO
Volume 22, Number 1, 2008
inhibition of the prenylated protein methyltransferase
[7,8]. It is conceivable, therefore, that the methylated
and demethylated forms of prenylated proteins may
be variously preferred for functional interactions by
different protein targets, thus rendering PPMTase and
PMPMEase very important moderators of prenylated
protein function.
OPs-sensitive, prenylation-dependent enzyme whose
substrates are large proteins of diverse structures, re-
spectively.
MATERIALS AND METHODS
Materials
The C-terminal cysteine residue with the
S-conjugated isoprenyl group is structurally specific
for interaction with other protein targets. For example,
unlike L-AFC, farnesol, farnesal, and farnesylic acid
are ineffective inhibitors of basal or receptor-mediated
binding of GTP-γ-S to receptors on HL-60 granulo-
cyte membranes [9]. This functional specificity may
be a product of coevolution that could have extended
to include interactions of the prenylated proteins and
PC analogs to PMPMEase. Rat liver supernatant and
brain membranes containing PMPMEase were previ-
ously found to hydrolyze prenylated substrates with
Michaelis–Menten constants of 5 and 25 µM, respec-
tively [10]. Using such prenylated cysteine ester sub-
strates to screen chromatographic fractions, it would
be possible to selectively enrich endogenous esterases
that metabolize prenylated proteins.
Serine esterases have active site serine, histidine,
and glutamate residues that form the catalytic triad in-
volved in bond hydrolysis. During catalysis, the active
site serine is temporarily acylated; the rapid hydrolysis
of the acyl-enzyme intermediate ensures the contin-
uous activity of the enzyme [11]. This mechanism is
exploited by other compounds that, acting as pseudo-
substrates for various hydrolytic enzymes, react with
the serine residue resulting in very slow recovery rates
with the net effect being that the covalently modified
enzyme molecules are unable to further bind and hy-
drolyze substrates [11]. Organophosphorus pesticides
(OPs) [11–13], lactones [14], sulfonyl fluorides [15], and
chloromethylketones [16] inhibit various serine hydro-
lases by this mechanism. In previous studies, preny-
lated methylated protein methyl esterase from rat liver
supernatant and brain membranes was inhibited by
OPs [10]. Similarly, a prenylation specific esterase from
bovine rod outer segment membranes was inhibited by
ebelactone B [14].
Porcine liver was obtained from Bradley’s
Country Stores (Tallahassee, FL) within 1 h of
slaughter and frozen until required for enzyme
preparation. Diethylaminoethyl (DEAE) sepharose,
phenyl sepharose, chelating sepharose, cibacron
blue sepharose, trans,trans-farnesol, benzil, and hy-
drocinnamoyl chloride were purchased from Sigma-
Aldrich (St. Louis, MO). Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) gels
were from Fisher Scientific (Suwannee, GA).
Synthesis of
Hydrocinnamoyl-farnesyl-L-cysteine
Methyl Ester
Farnesyl-L-cysteine methyl ester (FCM) was syn-
thesized as previously described [17]. The FCM (1 g)
was dissolved in approximately 100 mL of acetone fol-
lowed by the addition of triethylamine (TEA, 1 mL)
and 1 g of hydrocinnamoyl chloride (Scheme 1). This
was stirred for 2 h at room temperature. The solvent
was removed under reduced pressure; the residue was
dissolved in 150 mL of ethylacetate and washed three
times with water. The ethylacetate layer was dried with
anhydrous sodium sulfate and filtered, and the ethy-
lacetate was removed under reduced pressure. The
residue was purified by silica gel chromatography to
obtain hydrocinnamoyl-farnesyl-L-cysteine methyl es-
ter (HCFCM). The purified HCFCM was then ana-
lyzed by electron spray ionization mass spectrometry
(ESI-MS).
Enzyme Preparation and Assay
The supernatant fraction was prepared from 100 g
of porcine liver as previously described [10]. Assay
for PMPMEase activity was conducted using 1 mM
of either benzoyl-Gly-farnesyl-L-cysteine methyl es-
ter (BzGFCM) or HCFCM substrate as previously de-
scribed [10]. The supernatant was kept on ice and used
for the purification of PMPMEase.
In the current study, a carboxymethylated PC ana-
log substrate assay was used to purify an esterase
from porcine liver supernatant to apparent homogene-
ity. Tandem mass spectrometric analysis of the tryptic
peptide digest and database Mascot search identified
the enzyme as Sus scrofa carboxylesterase precursor. Its
features that include the active site serine hydrolase
catalytic triad of amino acids, the endoplasmic retic-
ulum retention signal, and the large, flexible and hy-
drophobic active site of the highly homologous human
form are consistent with the predicted properties of an
PMPMEase Purification
PMPMEase was purified from porcine liver su-
pernatant using a succession of different purification
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PRENYLATED METHYLATED PROTEIN METHYL ESTERASE
53
SCHEME 1. Synthesis of HCFCM and HCFC. HCFCM was synthesized from FCM and hydrocinnamoyl chloride as described
in the methods section. This was then purified over silica gel, eluting with a gradient of hexane and acetone. Also shown is the
hydrolysis of HCFCM to HCFC by PMPMEase.
media. Ice-cold porcine liver supernatant (8.8 g of pro-
tein in 1000 mL) was loaded onto a DEAE ion-exchange
column (4.8 I.D. × 60 cm) that had previously been
equilibrated with 20 mM Tris–HCl (pH 7.4) containing
0.1% Triton X-100 (buffer A) at a flow rate of 5 mL/min.
This was followed by washing with buffer A until the
UV absorbance at 280 nm subsided to the preloading
values. The bound enzyme was then eluted from the
column using a linear gradient from 0 to 1 M NaCl in
buffer A over 2 h. Fractions (4.5 mL) were collected,
and aliquots were assayed for enzyme activity using
HCFCM as substrate. Fractions with enzymatic activ-
ity (fractions 121–310) were combined and subjected to
further purification by hydrophobic interaction chro-
matography (HIC) on a column of phenyl sepharose.
The pooled fractions from the DEAE column were sup-
plemented with NaCl to a concentration of 1 M and
then loaded at a flow rate of 1.5 mL/min onto the HIC
column (2.5 cm I.D. × 30 cm) that had been preequili-
brated with buffer A containing 1 M NaCl. After load-
ing, the column was washed with the same buffer and
the enzyme was eluted with a linear gradient from 1 to
0 M NaCl in the same buffer. Fractions (4.5 mL) were
collected, and aliquots of the fractions were analyzed
for enzyme activity. The fractions with enzymatic ac-
tivity were combined and subjected to further purifi-
cation on Ni2+-charged immobilized metal ion affinity
chromatography (Ni-IMAC). The chelating resin was
charged with Ni2+ ions and equilibrated with buffer
A. The pooled enzyme containing fractions from the
HIC step was applied to the Ni-IMAC column at a
flow rate of 0.5 mL/min and washed with buffer A.
Elution was conducted with an increasing gradient of
histidine from 0 to 50 mM in the same buffer. Aliquots
of the 4.5 mL fractions were assayed for PMPMEase ac-
tivity. The combined active fractions were further pu-
rified on a Q-sepharose column (1 cm I.D. × 15 cm).
The binding and elution was conducted with the same
buffers as in the DEAE purification step except that
the required volumes were significantly less. Fractions
(4.5 mL) were collected, and aliquots were assayed for
enzyme activity. Fractions with enzyme activity were
combined and subjected to gel filtration chromatogra-
phy on a Superdex 200 column (2 cm I.D. × 90 cm) that
was preequilibrated and eluted with buffer A at a flow
rate of 2.5 mL/min. Enzyme-containing fractions were
further purified by Cu2+-charged immobilized metal
ion affinity chromatography (Cu-IMAC), loading and
washing in buffer A and eluting with a 0 to 50 mM gra-
dient of histidine in buffer A. The fractions containing
PMPMEase activity were combined and applied onto a
Cibacron blue 3GA column (1 cm I.D. × 15 cm) that had
been pre-equilibrated with buffer A. The flow-through
contained virtually all of the PMPMEase enzymatic
activity.
Protein Assays
The total protein concentration in various samples
was measured using the bicinchoninic acid (BCA) pro-
tein assay reagent kit (Pierce, Rockford, IL) according to
the supplier’s procedures. Bovine serum albumin was
used as the standard.
SDS-PAGE Analysis
Aliquots (50 µL) of all protein samples were com-
bined with 50 µL of SDS-PAGE sample buffer and
boiled for 5 min. The samples were then separated on
4–12% gradient gels, and the protein bands were visu-
alized by EZBlue coomassie staining (Sigma Chemical
Co., St. Louis MO). SDS-PAGE molecular weight mark-
ers were used to calculate the molecular weight of the
purified protein.
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OBOH AND LAMANGO
Volume 22, Number 1, 2008
given the S-farnesyl tail, HCFCM displayed compara-
ble properties to BzGFCM with respect to the kinetics
of hydrolysis by enzyme preparations.
Protein Sequence Analysis
Purified PMPMEase from the last purification step
(Cibacron Blue) was separated by SDS-PAGE (4%–
12% gradient gels) electrophoresis followed by EZBlue
coomassie staining. The protein bands were excised
and submitted to Protana Analytical Services (Toronto,
Ontario, Canada) for sequence analysis and protein
identification. The protein bands were subjected to in-
gel trypsin digestion, peptide elution, and liquid chro-
matography coupled with tandem mass spectrometric
(LC-MS/MS) analysis. The masses of the peptides were
detected, and the peptides were further fragmented to
obtain their individual sequences. The obtained pep-
tide masses and sequences were then used in Mascot
database searches for protein identification.
Purification of Liver PMPMEase
Various chromatographic media and conditions
were used to sequentially purify porcine liver PMP-
MEase. As shown in Figure 2, PMPMEase bound to the
different chromatographic media and was successfully
eluted according to the procedures stated in the meth-
ods section. The enzyme did not bind to the Cibacron
blue that was used in the last chromatographic step.
The Cibacron blue did, however, appear to bind to some
of the contaminating proteins as the enzyme flowed
through. As shown in Table 1, the specific activity in-
creased from 0.4 to 5.29 nmol of HCFCM hydrolyzed
per min per mg of protein. This coincided with an over
13-fold enrichment of the PMPMEase activity. More
than 80% of the enzyme activity was lost during the
first purification step that involved ion exchange on
DEAE sepharose (Table 1). SDS-PAGE protein analysis
of samples from the different chromatographic steps re-
vealed a successive elimination of contaminating pro-
tein bands that culminated in a single band of 57 kDa
observed in the unbound fraction from the Cibacron
blue step (Figure 3). This shows that although the en-
zyme did not bind to the Cibacron blue medium, it did
indeed trap the remaining contaminating proteins from
the Cu-IMAC column.
Effect of Serine Hydrolase Inhibitors
on PMPMEase Activity
Representative compounds from various classes
of chemicals known to inactivate serine hydrolases
were tested against porcine liver PMPMEase. These
included ebelactones, carbamates, chloromethylke-
tones, phenylmethylsulfonyl fluoride (PMSF), and
organophosphorus compounds. The compounds (in
5 µL of dimethylformamide, 1 and 0.1 mM) were added
to the enzyme (in 90 µL of 100 mM Tris–HCl buffer, pH
7.4) and preincubated for 20 min prior to the addition
of the HCFCM substrate to a concentration of 1 mM.
This was followed by further incubation and analysis
as described in the “enzyme preparation and assay”
section.
Protein Sequence Analysis
A single esterase of the serine hydrolase family
was detected when SDS-PAGE gel slices of the pu-
rified PMPMEase were trypsinized and the peptide
fragments were analyzed by LC-MS/MS followed by
Mascot searches of sequence databases. As shown in
Table 2, the masses and sequences of 17 tryptic-digest
peptides were detected, which are identical to por-
tions of the 62.3 kDa Sus scrofa carboxylesterase (pI
of 5.69) or its precursor (pI of 5.62). The sequences
of the 17 detected peptides amounted to 242 amino
acids or 43% of the Sus scrofa carboxylesterase se-
quence (accession number NP 999411). Three peptides
(FWANFAR, LGIWGFFSTGDEHSR and IPLQFSED-
CLYLNIYTPADLTK) matched the Mus musculus es-
terase 22, an enzyme with similar sequence, molec-
ular weight and pI characteristics as the Sus scrofa
carboxylesterase. A protein identified as hypothetical
protein from “Pongo pygmeus” matched the sequences
of 10 of the peptides accounting for 22% of the en-
tire 60-kDa protein. Both the Mus musculus esterase 22
and the Pongo pygmaeus hypothetical protein appear
RESULTS
Synthesis of
Hydrocinnamoyl-farnesyl-L-cysteine
Methyl Ester
The reaction of FCM with hydrocinnamoyl chlo-
ride resulted in hydrocinamyl-S-farnesyl-L-cysteine
methyl ester (HCFCM) as a waxy yellowish solid.
When this was analyzed by ESI-MS, a single peak
with a molecular weight of 494.26, corresponding to
the sodium ion adduct of HCFCM, was observed
(Figure 1A). When HCFCM (1 mM) was incubated with
porcine liver supernatant, a time-dependent hydrolysis
of the methyl ester to a single UV-absorbing product
peak that coeluted with the free acid (hydrocinamyl-
farnesyl-L-cysteine, HCFC) was observed (Figures 1B
and C). This indicates the hydrolysis of the ester bond of
HCFCM to form HCFC. Hydrolysis was not observed
in control incubations lacking enzyme. As expected
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PRENYLATED METHYLATED PROTEIN METHYL ESTERASE
55
FIGURE 1. Mass spectrometry analysis of HCFCM (A). HCFCM (1 mM solution in methanol) was analyzed by electron spray ionization
(ESI) mass spectrometry revealing a single distinct peak with an apparent relative molecular mass of 471.70. This corresponds to the calculated
molecular mass of 471.70 (494.261 less the atomic mass for Na of 22.99). Reserpine was included in the analysis as an internal standard. Time-
dependent demethylation of HCFCM to HCFC by pig liver supernatant PMPMEase (B and C). Porcine liver supernatant (0.4 mg of protein)
was incubated with HCFCM (1 mM) in 100 mM Tris–HCl, pH 7.4 for the indicated times (right side of each chromatogram, panel A). Reactions
were stopped with methanol and analyzed as indicated in the methods section. The results are the means ( SEM, n = 3. N.B. The error bars are
obscured by the symbols).
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OBOH AND LAMANGO
Volume 22, Number 1, 2008
to be house mouse and orangutan species variants of
the porcine carboxylesterase. Owing to the high sensi-
tivity of LC-MS/MS analysis, other peptide sequences
were detected that match 14 other proteins in sequence
databases. Of these, none is an esterase, the only other
detected hydrolase being aminoacylase 1 that hydroly-
ses acetyl groups from N-acetylated proteins [18].
When the Sus scrofa amino acid sequence was
used to conduct a BLAST database search, several car-
boxylesterases from different mammalian species and
tissues were identified that show a 71%–80% iden-
tity and 83%–89% similarity to the Sus scrofa car-
boxylesterase 3 (Table 3). These have been reported to
catalyze such reactions as the hydrolysis of triacylglyc-
erols, cholesterol ester, cocaine, retinyl ester, as well
as acyltransferase reactions. Until they are tested with
prenylated ester substrates, the possibility that these
highly homologous enzymes may be species/tissue
variants of the porcine liver PMPMEase that might
have coevolved with the ubiquitous prenylated pro-
teins will remain unclear.
Hydrolysis of Farnesyl-L-cysteine Methyl
Esters by Purified PMPMEase
Purified PMPMEase hydrolyzed both BzGFCM
and HCFCM in a concentration-dependent manner
(Figure 4). Michaelis–Menten analysis of the sub-
strate hydrolysis revealed Km values of 33 4 and
25 4 µM and Vmax values of 4.51 0.28 and 6.80 0.51
nmol/min/mg of protein for BzGFCM and HCFCM,
respectively. The Km for BzGFCM using the purified
porcine liver enzyme is about six-fold higher than that
previously obtained using rat liver supernatant but
comparable to that obtained with rat brain membrane
bound enzyme [10].
FIGURE 2. Chromatographic purification of PMPMEase. All purifi-
cation steps were conducted in 20 mM Tris–HCl, pH 7.4 containing
0.1% Triton X-100 as buffer A. All fractions were assayed for PMP-
MEase activity using HCFCM as the substrate. Porcine liver super-
natant (8.8 g, 1000 mL) was applied to a DEAE-sepharose column
and washed in buffer A. Bound proteins were eluted by an increas-
ing linear gradient of 0 to 1 M NaCl in the buffer A. The PMPMEase-
containing fractions from the DEAE step were applied to an HIC
column in buffer A containing 1 M NaCl. Elution was achieved using
a decreasing linear gradient of 1 to 0 M NaCl. The enzyme fractions
from the HIC column were applied to the Ni-IMAC column. The
enzyme was eluted with a linear gradient of 0 to 50 mM histidine
in buffer A. The enzyme fractions were applied to a Q-sepharose
column and eluted with a linear gradient of 0 to 1 M NaCl in buffer
A. Fractions from the Q-sepharose column were further purified on
a Superdex 200 gel-filtration column, eluting with 1 NaCl buffer A.
The enzyme fractions were further purified on a Cu-IMAC column,
eluting as in the case of Ni-IMAC. The resulting enzyme was then
applied to a Cibacron blue column and eluted as described in the
methods section.
Inhibition of Purified PMPMEase by Serine
Hydrolase Inhibitors
The active site hydroxyl group of the catalytic ser-
ine residue of serine hydrolases is often the target
of various compounds that covalently modify it re-
sulting in the loss of enzymatic activity [13,19]. The
compounds, thus acting as pseudosubstrates, react
with the enzyme but unlike the normal carboxylester
substrates, the pseudosubstrates are not converted to
products. The longer duration of active site occu-
pancy prevents access by the substrates thereby in-
hibiting the enzyme. Of the compounds tested against
the purified enzyme, ebelactone B, paraoxon, and
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PRENYLATED METHYLATED PROTEIN METHYL ESTERASE
57
TABLE 1. Purification of Porcine Liver PMPMEase
Total Activity
(nmol/min)
Total Protein
(mg)
Specific Activity
(nmol/min/mg)
Purification
Factor
Chromatographic Step
Yield (%)
Supernatant
Diethylaminoethyl
Hydrophobic interaction chromatography (HIC)
Ni2+-charged immobilized metal ion affinity
chromatography
3485
704
8805
3113
484
0.40
0.23
1.38
1.41
1.00
0.57
3.48
3.57
20
19
11
667
374a
265
Q-sepharose
Gel-filtration
444
221
150
220
75
60
2.02
2.96
2.49
5.09
7.47
6.28
13
6
4
Cu2+-charged immobilized metal ion affinity
chromatography (Cu-IMAC)
Cibacron blue
127
24
5.29
13.23
3.6
Aliquots of the pooled enzyme containing fractions from each of thechromatographic steps were assayed for the total enzyme activity and total protein. The values
obtained were then used to calculate the specificactivities, purification factors, and yields for the chromatographic steps.
a Lower enzymatic activity than in the proceeding step possibly due to leached Ni2+ ions interfering with enzyme activity.
more than 80% of the enzyme at 1 mM concentrations.
The other chloromethyl ketones inhibited about 45% of
the activity at concentrations of 1 mM. The carbamate
compounds did not inhibit the PMPMEase at the tested
concentrations.
DISCUSSION
Readily reversible secondary modifications such
as kinase-catalyzed phosphorylation and phosphatase-
catalyzed dephosphorylation reactions strongly influ-
ence both the conformations and the functions of var-
ious proteins. Such effects are only as impacting as
the relative activity levels of the counteracting en-
zymes that metabolize them. In prenylated protein
metabolism, two enzymes catalyze counteracting re-
actions in the final step of the pathway. An esteri-
fication reaction is counteracted by an esterase [2],
thus ensuring a healthy physiological balance of the
C-terminal uncharged methyl ester prenylated pro-
teins and the charged carboxylate isoforms. The charge
states of the carboxyl ends of the prenylated proteins
may determine both their structures and the nature
of the interactions with other proteins that are depen-
dent on the polyisoprenyl moiety and vicinal groups.
A polyisoprenyl-binding pocket was demonstrated on
FIGURE 3. SDS-PAGE analysis. (A) SDS-PAGE sample buffer
the Rho dissociation inhibitor [6] that strongly indi-
cates the importance of the farnesyl or geranylger-
anyl moiety and indeed of the prenylation pathway
changes on protein–protein interactions. Furthermore,
a strong influence on physiological well being was
demonstrated with mice deficient in isoprenyl car-
boxylmethyl transferase (PPMTase) which, unable to
methylate prenylated protein substrates, did not sur-
vive through midgestation [20]. Although this could, in
some circumstances, be synonymous with an overac-
tive PMPMEase that would ensure a lower proportion
(25 µL) was added to 25 µL aliquots of the combined fractions with
PMPMEase activity from each of the chromatographic steps, boiled
and separated on 4%–12% gradient SDS-PAGE gels followed by
EZBlue coomassie staining. The Rf values for the molecular weight
standards were determined and used to generate the calibration plot
(panel B) used to determine the molecular weight of the single protein
band from the Cibacron blue lane.
PMSF completely inhibited the purified enzyme at
1 mM concentrations (Table 4). Ebelactone A, L-I-4-
tosylamino-2-phenylethyl chloromethyl ketone, and
tosyl-L-phenylalanine chloromethyl ketone inhibited
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TABLE 2. Peptides Detected by In-Gel Trypsin Digestion of the Purified Enzyme Followed by LC-MS/MS Analysis
Peptides Detected
Calculated Molecular Mass
Observed (M/Z)
FWANFAR
TATSLLWK
QIAVLAGCK
910.44
918.52
958.53
456.60
460.33, 919.41
480.46
TTTSAVFVHCLR
QKSEDELLDLTLK
1390.70
1530.81
1599.81
1708.65
1747.91
1957.00
2190.14
2519.30
1063.52
1442.81
1506.74
1847.02
1259.59
2924.65
697.18
766.70
801.08
855.33
875.11
979.70
FAPPQPAEPWSFVK
LGIWGFFSTGDEHSR
LKGEEVAFWNDLLSK
SYPIANIPEELTPVATDK
TVIGDHGDEIFSVFGFPLLK
MPEEILAEKDFNTVPYIVGINK
YLGGTDDPVK
AISESGVALTAGLVR
GEEVAFWNDLLSK
ESHPFLPTVVDGVLLPK
GDAPEEEVSLSK
1096.01
841.25
1064.41, 533.51
722.96
754.69
924.73
630.93
976.37
YVSLEGLAQPVAVFLGVPFAKPPLGSLR
TABLE 3. Sequence Identity and Similarity of the Purified Enzyme to Other Esterases
Accession Number
Identity (%)
Similarity (%)
Reference
Sus scrofa CES3
Canis familiaris CESdD1
Homo sapiens CES hBr2
Felis catus CES1, mRNA
Bos taurus retinyl ester hydrolase (BREH1)
Macaca fascicularis CE
Homo sapiens CES1 (monocyte/macrophage)
Homo sapiens acyl CoA: cholesterol acyltransferase
Homo sapiens cholesterol ester hydrolase
Oryctololagus cuniculus liver carboxylesterase
Rattus norvegicus liver esterase (ES-10)
Mesocricetus auratus liver carboxylesterase precursor
Mus musculus triacylglycerol hydrolase
Homo sapiens CES hBr3, brain carboxylesterase
Mus musculus CES1
NP 999411
BAB60696
BAB85656
BAC75712
AAR14316
BAA24523
P23141
100
80
79
78
78
77
77
76
76
75
75
75
75
74
71
100
89
88
87
87
86
87
86
86
86
85
84
85
84
83
[23]
[23]
Hosokawa et al., unpublisheda
Miyazaki et al., unpublisheda
Wu et al., unpublisheda
Sone et al., unpublisheda
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
P23141
AAP20868
AAC39258
P16303
BAA05913
AAK58067
BAA84996
AAH26897
A BLAST search was conducted with the full amino acid sequence of the Sus scrofa carboxylesterase precursor protein. Several sequences were detected, all with
sequence identity and similarity to the query sequence of at least 71% and 83%, respectively. The proteins shown in the table were chosen for their reported substrates,
species and tissue source, and for their sequence comparisons to the Sus scrofa carboxylesterase.
a Available in the database with the respective sequences.
of prenylated and methylated proteins when compared
to the unmethylated counterparts, the significance of
PMPMEase on the physiological effects of prenylated
proteins is yet to be determined. Its purification and
identification is the very first step toward the under-
standing of its properties and how its levels of activity
may influence cellular and physiological events other
than the immediate hydrolysis of prenylated protein
methyl esters.
The tandem mass spectrometry analysis and iden-
tification of the enzyme as a Sus scrofa carboxylesterase
precursor is interesting in several respects. It shows
high-sequence identity and even higher sequence sim-
ilarity to various mammalian esterases that hydrolyze a
wide range of mainly hydrophobic substrates and have
molecular weights around 60 kDa [21–23]. These fea-
tures are similar to those of the purified PMPMEase,
which shows a molecular weight on SDS-PAGE of
about 57 kDa, and both the rat liver [10] and porcine
liver in this study show a high affinity for the hy-
drophobic PC analog ester substrates. The crystal struc-
ture of the human isoform, hCE1, reveals complexes
of homotrimers at equilibrium with the homohexam-
ers and the catalytic triad of Ser221–His468–Glu354 in-
side a deep hydrophobic pocket that has a small rigid
subsite and a large flexible region [24,25]. Purification-
induced dissociation of these multimeric complexes es-
pecially when high-salt concentrations were involved
might have resulted in monomeric, enzymatically in-
active conformations. This may thus explain the low
yields of the purified enzyme activity since separate
purifications of the enzyme with and without Triton
X-100 resulted in similar losses in activity following
the use of high-salt concentrations. The active site
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PRENYLATED METHYLATED PROTEIN METHYL ESTERASE
59
adaptive feature that could have evolved to accommo-
date the structural diversity of the polypeptide portions
of the prenylated protein substrates. These enzymes
have been cloned/isolated from a wide range of tis-
sues (see references in Table 4), a feature that is consis-
tent with the ubiquitous nature of prenylated proteins.
PMPMEase/Sus scrofa carboxylesterase, like the other
highly homologous carboxylesterases, has the HXEL
endoplasmic reticulum retention signal [28]. This re-
tention signal has been reported in the sister enzyme,
PPMTase/icmt, that catalyzes the forward prenylated
protein methylation reaction [29]. This colocalization
with the better characterized PPMTase/icmt further
supports the notion that the esterase isolated in this
study is indeed a prenylation pathway enzyme.
PMPMEase through its possible regulation of the
functions of various types of prenylated proteins may
exert profound effects on various intracellular events
and consequently on animal physiology. Putative sub-
strates include both heterotrimeric guanine nucleotide
binding proteins (G-proteins), monomeric G-proteins,
nuclear lamins, etc. [30]. These proteins mediate pro-
cesses ranging from neurotransmitter signaling, cy-
toskeletal, and intracellular transportation functions,
cell proliferation, differentiation, and apoptosis [30].
It could be inferred from these that aberrant levels
of PMPMEase activity would be expressed through
disease states such as cancers, neurodegenerative and
neuropsychiatric disorders. In this regard, the identifi-
cation of PMPMEase as a serine hydrolase is interesting
with respect to and consistent with its susceptibil-
ity to OPs pesticides, benzil, PMSF, chloromethylke-
tones, and ebelactones A and B. Exposure to OPs has
been widely reported to cause delayed Parkinson’s dis-
ease (PD)-like dyskinesias in cases that survive the
FIGURE 4. Michaelis–Menten kinetics (panel A) and double recip-
rocal plot (panel B) analysis of purified liver PMPMEase with prenyl-
L-cysteine analog substrates. Purified liver PMPMEase (5.262 µg) was
incubated with various concentrations of HCFCM ( ) or BzGFCM
◦
(
) substrates as described in the methods section. The results are
•
the means SEM of triplicate determinations.
characteristics are believed to be adaptations for bind-
ing the structurally diverse set of substrates such as
cocaine and heroin [24,26]. The enzyme has been de-
scribed as being promiscuous due to its large and flex-
ible active site that renders it the ability to hydrolyze a
wide variety of substrates [24,27]. The toxic substances,
narcotics, and pharmaceutical drugs and prodrugs that
the carboxylesterases hydrolyze are not endogenous
compounds. It is possible that the primary task of these
esterases is to metabolize prenylated proteins which are
indeed endogenous. The active site flexibility may be an
TABLE 4. Inhibition of Purified PMPMEase by Serine-Hydrolase Inhibitors
Residual Activity (% of Control, SEM, n = 3)
0.1∗/0.01 mM
Compounds Tested
1 mM
Asulam
Benzil
Benzyl carbamate
91.7 6.9
38.0 .43
113 6.3
∗69.4 .24
117 11
124
5
Ebelactone A
18.3 0.3
123
7
Ebelactone B
0
0
56.4 1.0
93.3 1.0
95.5 3.0
94.8 1.3
105 3.1
98.3 1.9
96.5 1.4
99.1 5.6
L-Leu chloromethyl ketone (CMK)
L-I-4-Tosylamino-2-phenylethyl CMK
N-Carbobenzyloxy-L-Phe CMK
N-(Methoxysuccinyl)-L-Ala-L-Ala-L-Pro-L-Val CMK
N-α-Tosyl-L-Lys CMK
Tosyl-L-Phe CMK
(3S)-1-Chloro-3-tosylamido-7-amino-2-heptanone
Paraoxon
60.4 1.9
18.0 2.1
57.0 2.0
61.8 2.0
98.6 7.3
13.9 1.1
73.6 1.2
0
0
0
0
0
0
Phenylmethylsulfonylfluride
79.5 1.8
Purified PMPMEase (10 µg) was incubated with 1, 0.1 (benzil only), and 0.01 mM concentrations of the indicated compounds for 20 min at 37◦C. HCFCM (1 mM)
was then added followed by further incubation and HPLC analysis.
J Biochem Molecular Toxicology DOI 10:1002/jbt

60
OBOH AND LAMANGO
Volume 22, Number 1, 2008
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