From the Departments of Pharmacology and Cancer
Biology and
Biochemistry, Duke University Medical Center,
Durham, North Carolina 27710 and the ¶ Department of Chemistry,
University of Illinois, Urbana, Illinois 61801
Received for publication, September 5, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Prenylated proteins contain either a 15-carbon
farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached
via a thioether bond to a cysteine residue at or near their C terminus. As prenylated proteins comprise up to 2% of the total protein in
eukaryotic cells, and the thioether bond is a stable modification, their degradation raises a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that cleaves prenylcysteines to cysteine and an unidentified isoprenoid product. Here we show that the isoprenoid product of prenylcysteine lyase is the
C-1 aldehyde of the isoprenoid moiety (farnesal in the case of
C-15). The enzyme requires molecular oxygen as a cosubstrate and
utilizes a noncovalently bound flavin cofactor in an
NAD(P)H-independent manner. Additionally, a stoichiometric amount of
hydrogen peroxide is produced during the reaction. These surprising
findings indicate that prenylcysteine lyase utilizes a novel oxidative
mechanism to cleave thioether bonds and provide insight into the unique role this enzyme plays in the cellular metabolism of prenylcysteines.
Covalent modifications by lipids play important roles in the
subcellular localization and biological activity of a multitude of
proteins in eukaryotic cells (1). One major class of lipid modification
is termed prenylation, in which either a 15-carbon farnesyl or
20-carbon geranylgeranyl isoprenoid is covalently attached via a
thioether bond to cysteine residue(s) at or near the C terminus of the
protein (2, 3). The majority of prenylated proteins contain the
so-called CaaX motif, which is defined by the
presence of a cysteine residue fourth from the C terminus of the
protein to which the isoprenoid is attached (4). CaaX proteins are subject to additional modifications after prenylation in
that the three C-terminal residues are proteolytically removed, and the
new C-terminal prenylcysteine is subject to carboxyl methylation (4,
5).
Prenylation is a stable modification, and these proteins can comprise
up to 2% of total cellular protein (6, 7). The quantity of prenylated
protein in cells, as well as the stability of the modification, raises
a metabolic challenge to the cell in the disposal of prenylcysteines
resulting from the normal turnover of these proteins.
S-Acylation, another lipid post-translational modification,
raises a similar metabolic challenge to the cell. An enzyme termed
palmitoyl protein thioesterase removes the palmitoyl group from
thiol-containing compounds arising from the degradation of
palmitoylated proteins (8), and this enzyme has been implicated in a
neurodegenerative lysosomal storage disorder, infantile neuroceroid lipofuscinosis (9). These findings in particular have highlighted an
important role for enzymes involved in the metabolism of lipidated proteins.
In a continuing effort to understand the metabolic fate of
prenylcysteines produced during turnover of prenylated proteins, we
identified and cloned a lysosomal enzyme that catalyzes the degradation
of prenylcysteines (10, 11). This enzyme, dubbed prenylcysteine lyase
(PCLase),1 degrades
prenylcysteines to yield free cysteine and an unidentified isoprenoid
product (10). The production of free cysteine (i.e. containing a reduced sulfhydryl) and the lack of a NAD(P)H requirement suggested that PCLase utilizes a mechanism distinct from enzymes such
as the carbon-sulfur Materials--
Farnesal was synthesized with modification using
farnesol as described (12). [35S]Farnesylcysteine (FC)
and [1-14C]FC were synthesized as described (10). Native
PCLase was purified from bovine brain as described (10).
Diphenyleneiodonium chloride (DPI) was obtained from Sigma.
7-Diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin was obtained
from Molecular Probes Inc.
Production and Purification of Recombinant
His6-PCLase--
Recombinant PCLase was produced by
infection of Sf9 cells with a recombinant baculovirus containing
the PCLase cDNA using a variation of the previously published
procedure (11). Specifically, histidine-tagged PCLase was produced by
insertion of six histidines between residues 496 and 497 in the
C-terminal region of PCLase using the Gene Editor kit (Promega)
following the manufacturer's instructions; the previously described
pFastBac/PCLase construct (11) was used as a template for mutagenesis.
Recombinant baculovirus containing this His6-PCLase variant
was produced using the Bac-to-Bac system (Life Technologies, Inc.) and
was used to infect Sf9 cells as described previously (11) except
that serum-free media was used, and the cells were harvested at
48 h post-infection.
Recombinant His6-PCLase was purified from extracts of
infected Sf9 cells by a combination of nickel-affinity and
anion-exchange chromatography. The cell pellet from a 4-liter
culture of infected Sf9 cells was resuspended in 40 ml of 50 mM Tris-Cl, pH 7.7, 0.2 mM EGTA, 0.2 mM EDTA, containing a mixture of protease inhibitors (13).
Cells were disrupted, and the homogenate was centrifuged at
100,000 × g for 1 h. PCLase was extracted from
the resulting pellet with 20 ml of 50 mM Tris-Cl, pH 7.7, containing 100 mM NaCl, 0.5% Triton X-100, and the
protease inhibitor mixture, followed by centrifugation at 100,000 × g for 1 h. The supernatant was loaded onto a 1-ml
column of nickel-nitrilotriacetic acid resin (Qiagen), the column was
washed with 5 ml of 50 mM Tris-Cl, pH 7.7, containing 100 mM NaCl, 0.2%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),
and 5 mM imidazole, and PCLase eluted with a 40-ml linear gradient of imidazole (5-500 mM) in the above buffer.
Fractions containing PCLase activity were pooled, dialyzed against 20 mM Tris-Cl, pH 7.7, containing 0.2% CHAPS (Buffer A), and
the dialysate loaded onto a Mono Q HR 5/5 column equilibrated with
Buffer A. PCLase was eluted with a 20-ml linear gradient of NaCl from 0 to 500 mM in Buffer A. Fractions containing PCLase were
pooled, dialyzed as above, flash-frozen in aliquots, and stored at
Identification of the Isoprenoid Product of the PCLase
Reaction--
Purified bovine PCLase (120 ng) was incubated with 6.66 µM [14C]FC (~50 mCi/mmol) in 15 mM Tris-Cl, pH 7.7, 15% glycerol, 0.02% CHAPS, and 10 mM Zwittergent 3-14 for 30 min at 37 °C. In some reactions, semicarbazide was added to a final concentration of 40 mM after the enzymatic reaction, and the incubation was
continued for 30 min at 37 °C. Prior to HPLC analysis, 10 nmol of
unlabeled farnesylcysteine, farnesal, and the semicarbazide adduct of
farnesal were added to provide internal standards. Samples were
chromatographed on a C18 reverse-phase column with a 45-min
gradient from 10 to 100% acetonitrile containing 0.1% triflouroacetic
acid. Radioactivity was quantified by scintillation spectroscopy. The
elution positions of the isoprenoid standards were determined by
monitoring absorbance at 210 nm.
Determination of the Oxygen Requirement of PCLase--
PCLase
(300 nM) and reaction buffer (50 mM Tris-Cl, pH
7.7, containing 5 µM [35S]FC) were
deoxygenated by bubbling argon through the solutions. The solutions
were then mixed and incubated at 37 °C under argon. Aliquots (20 µl) were removed and quenched with the solvent used for thin-layer
chromatography (n-propanol:NH4OH:H2O
(6:3:1); 10 µl) at indicated times. At the time indicated in Fig. 2,
the reaction chamber was opened to air, and additional aliquots were
taken and reactions quenched as described above. Accumulation of the product [35S]cysteine was analyzed by thin-layer
chromatography as described (10).
Identification of the Flavin Moiety in PCLase--
The visible
spectrum of a 12 µM solution of PCLase in 20 mM Tris-HCl, pH 7.7, 0.2% CHAPS was recorded on a Hewlett
Packard diode-array spectrophotometer. The spectrum of the buffer alone was also determined and was subtracted from the enzyme spectrum. The
identification of the flavin as flavin adenine dinucleotide (FAD) was performed as described (14). Briefly, PCLase (240 µg in 100 µl) was denatured by addition of an equal volume of 20%
trichloroacetic acid and incubation for 15 min on ice. The sample was
then centrifuged, and the supernatant divided into two equal portions,
with one-half neutralized immediately by addition of 25 µl of 4 M K2HPO4, followed by determination
of its fluorescence (
Analysis of the inhibition of PCLase by DPI was performed by adding
increasing concentrations of DPI (see Fig. 3C) to PCLase (200 nM) in 50 mM Tris-HCl containing 5 µM [35S]FC. Reactions were conducted for 15 min at 37 °C, and product formation was determined by thin-layer
chromatography as described above.
PCLase Activity Assays--
PCLase activity was routinely
determined by following the conversion of [35S]FC
(~50,000 cpm/time point) to [35S]cysteine by thin-layer
chromatography as described (10). In the studies comparing cysteine and
hydrogen peroxide production, a fluorimetric assay of cysteine
production was also employed. This assay involved reacting the cysteine
formed during the reaction with the fluorogenic compound
7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin to yield a
fluorescent product. PCLase (300 nM) was mixed with saturating FC (100 µM) in 50 mM sodium
phosphate, pH 7.4, at 25 °C. At time intervals ranging to 30 min,
50-µl samples were removed and mixed with 50 µl of 0.2 mM 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin in the assay buffer. A control reaction without FC was processed in
parallel. The intensity of fluorescence was determined
( Measurement of Hydrogen Peroxide Production by
PCLase--
Hydrogen peroxide produced in the PCLase reaction was
measured using the Amplex Red kit (Molecular Probes). PCLase (10 ng/µl) was incubated at 23 °C in the buffer provided, which also
contained horseradish peroxidase and Amplex Red. After the base line
was established, the PCLase substrate FC was added to a final
concentration of 5 µM. The action of horseradish
peroxidase on Amplex Red in the presence of hydrogen peroxide converts
the Amplex Red into a fluorogenic product (16). Fluorescence analysis
of the reaction mixture was conducted ( Identification of the Isoprenoid Product of the PCLase
Reaction--
In the previous study, the PCLase action on FC was shown
to produce free cysteine and an unidentified hydrophobic moiety derived from the isoprenoid constituent (10). Because in that study the
isoprenoid product appeared to be less polar than farnesol (10), we
hypothesized that this product might be the isoprenoid aldehyde,
i.e. farnesal. To test this possibility, FC that
had 14C incorporated in the isoprenoid moiety was incubated
with purified bovine PCLase. Analysis of the reaction mixture by
reverse-phase HPLC revealed the expected loss of radioactivity in the
substrate FC because of digestion by PCLase, as well as the appearance
of a peak of radioactivity that comigrated with an authentic farnesal standard (Fig. 1, A and
B). When the reaction mixture was subjected to
derivatization with semicarbazide, a compound that forms an imine
adduct with aldehydes (17), the radiolabeled product of the reaction
now comigrated with the authentic farnesal-semicarbazone standard (Fig.
1C). Similar results were seen when reaction mixtures were
analyzed using a normal-phase thin-layer chromatography system (data
not shown). These data revealed that the isoprenoid product of the
PCLase reaction was the aldehyde of the isoprenoid and indicated an
oxidative type of mechanism for the enzyme.
Analysis of Molecular Oxygen Requirement for PCLase
Action--
The finding that PCLase produced an oxidized isoprenoid
suggested that the enzyme utilized molecular oxygen as a cosubstrate. To examine this possibility, the PCLase reaction was studied in the
absence and presence of oxygen. Solutions of the enzyme and assay
components were made anaerobic by degassing with argon, placed in a
sealed chamber, and aliquots were removed for analysis of product
formation. Essentially no product formation was detected under
anaerobic conditions (Fig. 2). However,
when the reaction chamber was opened to the atmosphere to allow oxygen
entry, product formation ensued in a linear fashion with a rate
constant of 0.25 min Detection of a Flavin Cofactor Associated with PCLase--
Many
enzymes that use oxygen also utilize a flavin and/or NAD(P)H as
cofactors (18). However, PCLase shows no NAD(P)H requirement for
catalysis (10). To investigate the possibility for other cofactors, the
visible spectrum of recombinant PCLase was measured. This spectroscopic
analysis revealed the presence of characteristic peaks of a flavoenzyme
at 360 and 454 nm (Fig. 3A)
(18).
To determine whether the apparent flavin cofactor was flavin
mononucleotide (FMN) or FAD, we took advantage of the facts that FAD
has a much lower intrinsic fluorescence than FMN because of the
To demonstrate that the bound FAD was required for catalysis, PCLase
was treated with DPI, a compound that blocks the reoxidation of FAD to
the reduced state (19). DPI treatment inhibited the PCLase reaction in
a dose-dependent fashion with an IC50 of ~100 µM (Fig. 3C). These results provided
compelling evidence that FAD participates in a catalytic manner in the
enzyme by cycling between its oxidized and reduced forms, a result
again completely consistent with an unusual oxidative mechanism for the enzyme.
Determination of Hydrogen Peroxide Production by PCLase--
Many
flavoenzymes generate hydrogen peroxide during catalysis by reduction
of oxygen (18). To determine whether PCLase produced hydrogen peroxide,
a peroxidase-linked assay was used that employed a nonfluorogenic
substrate (Amplex Red) that is converted to a fluorogenic compound by
horseradish peroxidase (HRP) in the presence of hydrogen peroxide (16).
Addition of the substrate FC to a mixture of PCLase in the presence of
HRP and Amplex Red resulted in a biphasic time course of hydrogen
peroxide formation (Fig. 4), the first
phase apparently being due to equilibration of the reaction, with the
second phase representing the steady state rate of hydrogen peroxide
production. The rate of the linear increase in hydrogen peroxide
formation in the second phase (kobs = 0.27 ± 0.02 min
Based on the data reported in this and the previous study (10), we
propose the mechanism for the PCLase reaction shown in Fig.
5. Although the mechanism depicted is not
the only one that could be drawn for PCLase, we believe that it is the
most likely mechanism, because it is the simplest one that can be
envisioned that accounts for all three products of this reaction,
i.e. free cysteine, the prenyl aldehyde, and
hydrogen peroxide. In this mechanism, the reaction is initiated by
FAD-mediated hydride abstraction from C-1 of the isoprenoid moiety,
producing a reduced flavin and an allylic (i.e.
resonance-stabilized) carbocation intermediate. Attack of a water
molecule on the carbocation results in formation of a hemithioacetal,
which then collapses to the prenyl aldehyde concomitant with C-S bond
breakage in which cysteine acts as the leaving group. Meanwhile the
hydride of the reduced flavin is transferred to molecular oxygen,
resulting in hydrogen peroxide formation and reoxidation of the
flavin.
In summary, this study provides evidence for a novel mechanism in the
catabolism of prenylcysteines by PCLase. These findings are
particularly intriguing in light of the fact that PCLase has essentially no homology to other known proteins (11). The uniqueness of
PCLase may be a result of uniqueness of its substrates; thioethers are
rare in mammalian organisms, and prenylcysteines are the only known
thioethers that contain an allylic carbon in the C-S bond. Characterization of structure-function relationships of PCLase are
likely to yield important findings as to how it achieves this type of
catalysis. These and other ongoing studies should provide insight into
this unusual enzyme that will allow a better understanding of the
cellular role of PCLase in the metabolism of prenylcysteines.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-lyase and cytochrome P450- and
flavin-containing monooxygenases known to cleave carbon-sulfur bonds
(10). Additionally, PCLase has essentially no sequence similarity to
known enzymes, suggesting that it might possess a novel mechanism for
carbon-sulfur bond cleavage (11). In the present study, the unusual
mechanism of the reaction catalyzed by PCLase was investigated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C until use.
ex = 450 nm;
em = 535 nm). The remaining half was incubated at 37 °C for 3 h in
the dark, neutralized as above, and its fluorescence was determined.
The ratio of FAD to protein was determined using the molar extinction
coefficient at 454 nm of 12.5 mM
1
cm
1 (15).
ex = 384 nm;
em = 470 nm) in a 100-µl
cuvette, and cysteine formed was calculated from a cysteine standard curve.
ex = 563 nM,
em = 587 nM).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Identification of the isoprenoid product of
PCLase. [1-14C]FC was incubated with buffer alone
(panel A) or with purified bovine PCLase (120 ng;
panels B and C) for 30 min at 37 °C. For the
reaction shown in panel C, semicarbazide (SCA; 40 mM) was added after the enzymatic reaction, and the
incubation was continued for an additional 30 min at 37 °C.
Reactions were terminated and analyzed by C18 reverse-phase
HPLC as described under "Experimental Procedures." Radioactivity in
each fraction was quantified by liquid scintillation spectroscopy. The
data shown are from a single experiment that is representative of three
such experiments.FCHO, farnesal.
1 (Fig. 2). These results indicated
that PCLase requires molecular oxygen for product formation and are
consistent with a reaction proceeding via an oxidative mechanism that
forms a prenyl aldehyde.
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Fig. 2.
Dependence of the PCLase reaction on
molecular oxygen. Deoxygenated PCLase (300 nM; see
"Experimental Procedures") was incubated in deoxygenated buffer
containing 5 µM [35S]FC in a sealed chamber
under argon. Aliquots were removed at the indicated time points,
reaction mixtures were resolved by thin-layer chromatography, and
cysteine product formation was visualized by fluorography and
quantified by liquid scintillation spectroscopy. The arrow
indicates the time when the chamber was opened. The data shown are the
average of duplicate determinations from a single experiment that is
representative of three such experiments.
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Fig. 3.
Identification of a flavin associated with
PCLase. Panel A, spectroscopic analysis of PCLase. The
visible spectrum of a 12 µM solution of PCLase is shown.
Panel B, fluorescence analysis of the flavin released from
PCLase. The supernatant obtained from acid denaturation of 240 µg of
recombinant PCLase was analyzed by fluorescence spectroscopy as
described under "Experimental Procedures." Panel C,
inhibition of PCLase by DPI. Purified recombinant PCLase was incubated
with the indicated concentration of DPI for 15 min at 37 °C, and
enzyme activity was then determined as described under "Experimental
Procedures." For all panels, the data shown are from
single experiments that are representative of at least two such
experiments.
-
stacking of the isoalloxazine ring system with the adenine ring of FAD
and that FAD can be converted to FMN by acid hydrolysis of the
diphosphate bond, thereby releasing this
-
interaction (14).
Therefore, PCLase was subjected to acid denaturation, and the denatured
enzyme was pelleted. The resultant supernatant was divided with
one-half being immediately neutralized and the other half incubated in
the acid for 3 h at 37 °C prior to neutralization. Analysis of
the fractions by fluorescence spectroscopy revealed an emission
signature consistent with the presence of a flavin (Fig.
3B). This fluorescence markedly increased in the sample subjected to acid hydrolysis (Fig. 3B), indicating the
identity of the flavin as being FAD. Furthermore, this experiment
indicated that the FAD cofactor was not covalently attached to the
enzyme, because it was released upon denaturation. In addition, the
absorption intensity at 454 nm corresponded to a ratio of FAD to
protein of 1.3:1 (data not shown; see "Experimental Procedures"),
indicating that the enzyme contained a stoichiometric amount of
FAD.
1) correlated well with the rate of cysteine
formation determined for aliquots of the same reaction mixture
(kobs = 0.18 ± 0.02 min
1,
indicating stoichiometric production of hydrogen peroxide during turnover by the enzyme.
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Fig. 4.
Determination of hydrogen peroxide production
by PCLase. Purified recombinant PCLase (10 ng/µl) was incubated
in the presence of horseradish peroxidase and Amplex Red reagent. The
substrate FC (5 µM) was added to initiate the reaction.
Hydrogen peroxide was detected as described under
"Experimental Procedures." The data shown are from a single
experiment that is representative of three such experiments.
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Fig. 5.
Proposed mechanism of PCLase. The
mechanism proposed for the reaction catalyzed by PCLase is shown.
R, the homogeranyl portion of the farnesyl unit.
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ACKNOWLEDGEMENTS |
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We thank Eric Furfine, Irwin Fridovitch, and Kendra Hightower for helpful discussions, Ted Meigs for comments on the manuscript, and Alan Embry for assistance with the figures.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants GM46372 (to P. J. C.) and GM13956 (to R. M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a National Institutes of Health postdoctoral fellowship.
** To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710-3686. Tel.: 919-613-8613; Fax: 919-613-8642; E-mail: casey006@mc.duke.edu.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.C000616200
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ABBREVIATIONS |
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The abbreviations used are: PCLase, prenylcysteine lyase; FC, farnesylcysteine; HPLC, high pressure liquid chromatography; DPI, diphenyleneiodonium chloride; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NAD(P)H, reduced nicotinamide adenine dinucleotide; HRP, horseradish peroxidase; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate.
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