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J Biol Chem, Vol. 274, Issue 30, 20772-20778, July 23, 1999
From the Laboratorium für Mikrobiologie, Fachbereich
Biologie, Philipps-Universität, D-35032 Marburg, Germany
The exchange of oxygen atoms between acetate,
glutaryl-CoA, and the catalytic glutamate residue in glutaconate
CoA-transferase from Acidaminococcus fermentans was
analyzed using [18O2]acetate together with
matrix-assisted laser desorption/ionization time of flight mass
spectrometry of an appropriate undecapeptide. The exchange reaction was
shown to be site-specific, reversible, and required both glutaryl-CoA
and [18O2]acetate. The observed exchange is
in agreement with the formation of a mixed anhydride intermediate
between the enzyme and acetate. In contrast, with a mutant enzyme,
which was converted to a thiol ester hydrolyase by replacement of the
catalytic glutamate residue by aspartate, no 18O uptake
from H218O into the carboxylate was detectable.
This result is in accord with a mechanism in which the carboxylate of
aspartate acts as a general base in activating a water molecule for
hydrolysis of the thiol ester intermediate. This mechanism is further
supported by the finding of a significant hydrolyase activity of the
wild-type enzyme using acetyl-CoA as substrate, whereas glutaryl-CoA is not hydrolyzed. The small acetate molecule in the substrate binding pocket may activate a water molecule for hydrolysis of the nearby enzyme-CoA thiol ester.
The strict anaerobic bacterium Acidaminococcus
fermentans is able to grow with glutamate as the sole source of
energy. The fermentation of glutamate proceeds via the hydroxyglutarate
pathway yielding ammonia, carbon dioxide, acetate, butyrate, and
hydrogen as products (1). With the exception of the oxidation of
glutamate to 2-oxoglutarate followed by reduction to
(R)-2-hydroxyglutarate, almost all other transformations in
this pathway are carried out on the CoA-ester level (2). Hence the
activation of (R)-2-hydroxyglutarate is a key step in this
metabolic pathway (3).
The catalytic action of CoA transferases has been suggested to proceed
via a mechanism outlined in Fig. 1 (6,
7). By nucleophilic attack of a glutamate residue of the enzyme at the carbonyl of the donor acetyl-CoA, a mixed anhydride between the enzyme
and acetate is formed. The transiently liberated CoAS
Oxygen Exchange between Acetate and the Catalytic Glutamate
Residue in Glutaconate CoA-transferase from Acidaminococcus
fermentans
IMPLICATIONS FOR THE MECHANISM OF CoA-ESTER HYDROLYSIS*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The transfer of coenzyme A from acetyl-CoA to
(R)-2-hydroxyglutarate (Equation 1) is catalyzed by
(E)-glutaconate:acetyl-CoA CoA-transferase (EC 2.8.3.12,
further referred to as glutaconate CoA-transferase), which can also use
propionate and glutarate as substrates. The enzyme has been purified,
and the two encoding genes have been cloned, sequenced, and
overexpressed in Escherichia coli. The hetero-octameric
protein (
(Eq. 1)
4
4) consists of two different subunits with molecular mass values of 35.5 kDa (-subunit) and 29 kDa (-subunit) (4, 5).
anion reattacks the glutamate carbonyl carbon to form the product acetate and an enzyme-CoA thiol ester. The second product,
(R)-2-hydroxyglutaryl-CoA, is formed by repetition of the
steps outlined above. It should be noted that during one catalytic
cycle, one oxygen atom is transferred from the acetate to the glutamate
residue. Consecutive turnovers lead to a complete equilibration of all
oxygen atoms involved in the reaction.
View larger version (12K):
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Fig. 1.
Reaction scheme of coenzyme A transfer by
glutaconate CoA-transferase. The oxygen exchanged between acetate,
glutaryl-CoA, and the catalytic glutamate residue of the enzyme
(E) is shown in bold letters. It should be noted that upon
several cycles of catalysis, both labeled oxygens are distributed
equally among all seven participating oxygen positions in the enzyme
and the substrates/products. To simplify the presentation, the
equilibrium reactions are shown in a single direction.
The catalytic glutamate residue in glutaconate CoA-transferase has been
identified as amino acid 54 of the smaller -subunit (Glu-54). The thiol ester between coenzyme A and the glutamate residue has been reduced with sodium boro[3H]hydride to
the corresponding alcohol, which was identified as 2-amino-5-hydroxy[5-3H]valeric acid within a tryptic
peptide (4, 5). The enzyme was crystallized, and its structure has been
solved recently at 2.55 Å resolution. The crystallographic data (8) as
well as sequence alignments (9) also confirmed that glutamate 54 is the catalytic residue in glutaconate CoA-transferase.
The important role of glutamate 54 has been demonstrated by
site-directed mutagenesis experiments (10). Whereas the replacement of
glutamate 54 by alanine (E54A) or asparagine
(E54N) completely abolished the transferase activity,
the glutamine (E54Q) mutant retained a remarkable
residual activity (1% of the wild type). By incubating this enzyme
with both substrates for 20 h at room temperature, the glutamine
was completely converted to glutamate yielding a fully active
CoA-transferase. Changing glutamate 54 to aspartate converted the
enzyme to a thiol ester hydrolyase (9). It has been suggested that the
missing methylene group in the side chain of aspartate as compared with
glutamate allows a water molecule to occupy the space between the
substrate and the carboxylate of aspartate 54. Activated by the
carboxylate of aspartate as the general base, this water molecule
should be able for a nucleophilic attack at the thiol ester carbonyl
carbon and cleavage of the thiol ester bond. Alternatively, a mechanism requiring the transient formation of a mixed anhydride between aspartate and the donor carboxylic acid might be possible.
The exchange of oxygen between the CoA donor, glutaryl-CoA, the
acceptor, [18O2]acetate, and the catalytic
glutamate residue could be followed by modern mass spectrometry
techniques (MALDI-TOF MS).1
The 18O label is shown to be specifically, reversibly, and
unequivocally located at glutamate 54. In contrast, the catalytic
aspartate residue in the E54D mutant, which acts as a
hydrolyase, contained no 18O when the reaction was carried
out in [18O]water.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Materials--
[18O]Water (purity 98%) was
purchased from Promochem (Wesel, Germany). TPCK-treated trypsin (EC
3.4.21.4), endoproteinase Asp-N (EC 3.4.24.33),
carboxypeptidase Y (EC 3.4.16.1), and coenzyme A (trilithium salt) were
from Roche Molecular Biochemicals. All other chemicals were of the
highest available grade and were purchased from Fluka (Buchs,
Switzerland), Sigma, or Merck. Glutaconate CoA-transferases (wild type
and E54D mutant) were purified from overproducing
E. coli strains as described earlier (5). The crystallization step, however, was replaced by chromatography on
Q-Sepharose. The molar concentrations of the hetero-octameric enzyme
were calculated with the molecular mass of the heterodimeric subunit
(65 kDa).
Synthesis of Acetyl-, Glutaryl-, and Propionyl-CoA-- The CoA thiol esters of acetic, glutaric, and propionic acid were prepared from the corresponding anhydrides and CoASH by the method of Simon and Shemin (11). The acyl-CoAs were desalted using Sep-PakTM C18 cartridges (Millipore).
Synthesis of [18O2]Acetate-- Ethyl acetimidate (1 mmol) was hydrolyzed for 2 h at 50 °C in 100 µl of [18O]water saturated with gaseous hydrochloric acid. Initially formed ethyl acetate was hydrolyzed in 100 µl of [18O]water adjusted to 1 M Na18OH with 3% (w/w) sodium amalgam at 50 °C for 16 h. The solution was adjusted to pH 8 with sodium hydrogen carbonate, lyophilized, and redissolved in [16O]water. The acetate concentration was determined enzymatically (12). The 18O content of the sodium acetate (99%) was determined by gas chromatography/mass spectrometry analysis of the methyl ester synthesized with diazomethane.
Kinetics of the Oxygen Exchange-- Glutaconate CoA-transferase (10 µg/ml) was added to a mixture containing 100 mM sodium phosphate, pH 7.0, 1 mM glutaryl-CoA, and 1 mM sodium [18O2]acetate. Before the addition of the enzyme and after various time intervals, aliquots were taken and stopped by the addition of 1 volume of 8 M guanidinium hydrochloride, pH 7.5. The samples were applied on LiChroCART columns (4 × 250 mm, 5 µm, Merck) equilibrated with 0.1% (v/v) trifluoroacetic acid. The CoA derivatives were separated by a linear gradient from 10 to 20% (v/v) acetonitrile for 20 min at 20 °C and monitored at 260 nm. The eluting CoA derivatives were collected and analyzed by MALDI-TOF MS (see below). The CoA derivatives were quantified using an internal calibration.
Labeling of Glutaconate CoA-transferase with [18O2]Acetate-- The enzyme (2 nmol) was incubated for 2 min at 37 °C in 200 µl of 100 mM sodium phosphate, pH 7.0, 1 mM glutaryl-CoA, and 1 mM sodium [18O2]acetate. The reaction was stopped by the addition of 1 volume of 8 M guanidinium hydrochloride, pH 7.5, and applied to reverse-phase HPLC as described. A second sample was incubated as described and separated from low molecular mass compounds by size exclusion chromatography using a Sephadex-G25 column (NAP-10, Amersham Pharmacia Biotech) equilibrated with 100 mM sodium phosphate, pH 7.0, and 100 mM [16O2]acetate. Glutaryl-CoA was added to the enzyme at a final concentration of 1 mM and incubated for another 2 min at 37 °C.
Chemical Hydrolysis of the Enzyme-CoA Thiol Ester Intermediate in
[18O]Water--
Glutaconate CoA-transferase (2 nmol) was
incubated for 1 min at 37 °C with 1 mM glutaryl-CoA in
200 µl of 100 mM sodium phosphate, pH 7.0. The reaction
products were applied to reverse-phase HPLC as described below. The
-subunit of glutaconate CoA-transferase was lyophilized, redissolved
in 25 µl of 4 M guanidinium hydrochloride, 400 mM Tris-HCl, pH 8.6, in [18O]water, and
incubated for 1 h at 50 °C. The mixture was diluted to 1 ml
with [16O]water, lyophilized, and reductively
carboxymethylated (13).
Incubation of Glutaconate CoA-transferase in
18O-Enriched Water--
The enzyme (2 nmol) was incubated
at 37 °C in 200 µl of 100 mM sodium phosphate, pH 7.0, 1 mM acetyl-CoA in the presence of 50% (v/v)
[18O]water for 5 (E54D) or 240 min (wild type).
Hydrophobic Interaction HPLC Separation of CoA Derivatives and Protein Subunits-- The samples were applied on a Supelcosil LC-3DP column (4.6 × 250 mm, Sigma) equilibrated with 0.1% (v/v) trifluoroacetic acid. CoA derivatives and enzyme subunits were eluted by a linear gradient from 0 to 84% (v/v) acetonitrile within 30 min and monitored at 280 nm. Fractions were collected manually and analyzed by MALDI-TOF MS (see below).
Matrix-assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS)-- Molecular mass values of glutaconate CoA-transferase subunits, peptides, and coenzyme A derivatives were determined using a Voyager RP workstation (Perseptive Biosystems). Prior to the preparation of the samples, acetonitrile was removed by centrifugation in vacuo. Protein solutions (1-5 µM) were mixed 1:1 with 110 mM sinapinic acid ((E)-3,5-dimethoxy-4-hydroxycinnamic acid) in 0.1% (v/v) trifluoroacetic acid, 67% (v/v) acetonitrile on the sample slide and air-dried. Peptide samples (0.5-3 µM) and CoA derivatives (5-20 µM) were applied on a thin layer of indole-2-carboxylic acid prepared from a solution of 300 mM indole-2-carboxylic acid in acetone. Details of the measurements are given in the figure legends.
Reductive Carboxymethylation and Tryptic Digestion of the
-Subunit--
Fractions containing the -subunit were
lyophilized, reduced with dithiothreitol, and carboxymethylated with
iodoacetate (13). Excess reagents were removed on Sephadex-G25 columns
(NAP-10) equilibrated with 50 mM ammonium acetate, pH 8.6, 10% (v/v) acetonitrile. The protein was digested for 16 h at
37 °C by 2% (w/w) TPCK-treated trypsin. The peptides were adjusted
to 4 M guanidinium hydrochloride, 0.1% (v/v)
trifluoroacetic acid and applied on a Supelcosil LC-318 column
(4.6 × 250 mm, Sigma) equilibrated with 0.1% (v/v)
trifluoroacetic acid. The column was developed by a linear gradient
from 0 to 42% (v/v) acetonitrile within 45 min. The eluting peptides
were monitored at 215 nm, collected manually, and identified by
MALDI-TOF MS.
Cleavage of the Tryptic Peptide 6 by Endoproteinase
Asp-N--
The tryptic peptide 6 was lyophilized, redissolved in
100 µl of 1 M guanidinium hydrochloride, 100 mM Tris-HCl, pH 7.5. Endoproteinase Asp-N (100 ng) was added, and the digestion was allowed to proceed for 16 h
at 37 °C. The resulting peptides were separated by reverse-phase HPLC as described above.
Carboxyl-terminal Sequencing--
The peptide 6A was
dissolved in 20 mM sodium acetate, pH 6.6, yielding a final
concentration of approximately 10 µM. The peptide
solution (2 µl) was mixed with 2 µl of carboxypeptidase Y (0.1 mg/ml) and incubated for 20 min at 25 °C. The reaction was stopped
by the addition of 2 µl of 10% (v/v) trifluoroacetic acid. The
individual molecular mass values of the generated peptides were
determined by MALDI-TOF MS (see above).
Determination of the Hydrolyase Activity of Glutaconate CoA-transferase-- The enzyme (0.77 nmol) was incubated at 25 °C in 1 ml of 100 mM potassium phosphate, pH 7.0, 100 mM NaCl, 1 mM 5,5'-dithio-bis-(2-nitrobenzoate), and 200 µM acyl-CoA in either the presence or the absence of 100 mM sodium acetate. The change in absorbance at 412 nm was recorded for 10 min.
Calculation of the 18O Content from Mass Spectra-- The natural isotopic distribution of large molecules such as acyl-CoAs and peptides was calculated solving the binomial equation for the isotopic distribution. This calculation was aided by the isotope pattern calculator provided by the University of Sheffield (UK) at the Sheffield ChemPuter web site (http://www.shef.ac.uk/chemistry/chemputer/isotopes.html). The 18O overlay was simulated by solving the binomial coefficient for different numbers of exchange sites. The distribution pattern for the enrichment of 18O is given by the general formula,
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(Eq. 2) |
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(Eq. 3) |
b,
b = 18O-enriched fraction of the total
oxygen of the sample, and n = number of possible sites
of oxygen exchange. Solved for different numbers of oxygen, it follows
that
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(Eq. 4) |
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(Eq. 5) |
The measured mass spectra were integrated, and the measured
distributions were fitted to simulated stick spectra minimizing the
least squares deviation between the intensities of measured and
simulated distributions. These evaluations were performed using the
"Solver" facilities provided by Excel 97. The experimental errors
of the measurements were determined by doubling the value of
2 for the nonlinear least square fits.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The time course of the oxygen exchange was investigated
with catalytic amounts (0.1 µM) of glutaconate
CoA-transferase and initial concentrations of 1 mM for
[18O2]acetate and glutaryl-CoA. At various
time intervals, samples were subjected to HPLC analysis and subsequent
MALDI-TOF MS of the CoA derivatives. As evident from the concentrations
of acetyl-CoA and glutaryl-CoA (Fig.
2C), a chemical equilibrium of
the reaction was reached after approximately 15 min with
Keq = 0.64 ± 0.04. This value is close to
0.77, which can be calculated from the pK values of
glutarate (pK1 = 4.34) and acetate
(pK = 4.75). In addition to acetyl- and glutaryl-CoA,
increasing amounts of free CoA were detected by HPLC, whereas neither
acetyl-CoA nor glutaryl-CoA exhibited significant rates of hydrolysis
in control experiments.
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As demonstrated in Fig. 2, the mass spectra of acetyl-CoA (Fig. 2A) and glutaryl-CoA (Fig. 2B) showed clearly resolved single isotopic peaks. The mass spectra were integrated, and the relative signal intensities for the isotopic distribution were calculated. Alignments of these distributions to simulations allowed the determination of the18O content of the acyl-CoAs. Minimizing the least squares deviation between simulation and measured data, the 18O content of the analytes could be calculated with an experimental error of less than 4% (see insets in Fig. 2, A and B). The isotopic equilibrium, however, was reached much more slowly than the chemical equilibrium after approximately 120 min. The 18O contents at equilibrium for acetyl-CoA (45 ± 3%) and glutaryl-CoA (41 ± 3%) agreed with the calculated value of 39.4%.
To analyze the incorporation of 18O into the catalytic
glutamate residue Glu-54 of glutaconate
CoA-transferase, 20 µM of enzyme was incubated with 1 mM unlabeled glutaryl-CoA and 1 mM
[18O2]acetate for 5 min, followed by HPLC
separation (Fig. 3). The 18O
label of the isolated acetyl-CoA (44 ± 3%) and glutaryl-CoA (43 ± 3%), determined by MALDI-TOF MS, indicated that an
isotopic equilibrium of the oxygen atoms participating in the reaction was reached.
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The separated subunits of glutaconate CoA-transferase were identified
by their molecular mass values obtained by MALDI-TOF MS. For the
-subunit a molecular mass of 35,573 ± 12 Da was determined in
agreement with the value of 35,568 Da predicted from the sequence (Fig.
4A). For the -subunit, a
molecular mass of 29,018 ± 18 Da (theoretical 29,017 Da) was
obtained. However, a second signal at m + 752 Da was found
(Fig. 4B). In incubations with glutaryl-CoA but without
acceptor carboxylate, the higher molecular mass signal was the only
visible one. The mass difference observed confirmed the covalent
binding of a CoA-molecule to the catalytic glutamate according to the
reaction scheme shown in Fig. 1.
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Neither the mass accuracy nor the resolution obtained in the spectra of
the -subunit was sufficient to determine 18O
uptake. Hence, the protein was reduced with dithiothreitol, carboxymethylated, and digested with trypsin. The
carboxymethylation was required to obtain the
Glu-54-containing peptide in good yields. It is well
established that a significant loss of Cys-containing peptides is
frequently observed if no chemical modification of the thiol group is
performed. The peptide containing the catalytic glutamate residue was
purified by reverse-phase HPLC on a Supelcosil LC-318 (4.6 × 250 mm, 5 µm). The monoisotopic molecular mass of that peptide
(2744.6 ± 1.5 Da) was, within the experimental range of accuracy,
identical to the expected mass of 2744.3 Da, but it showed a clearly
visible broadening of the isotopic distribution, which was only poorly
resolved (data not shown). Therefore, the peptide was digested for a
second time using endoproteinase Asp-N. As shown in Fig.
5, the undecapeptide containing amino
acids 48-DCHIIVESGLM-58 gave well resolved mass spectra. The
observed monoisotopic molecular mass of 1273.9 ± 0.5 Da was in
agreement with the theoretical mass of 1274.6 Da. The natural isotopic
distribution was clearly overlaid by an 18O distribution. A
closer analysis of the signal distribution revealed the presence of two
18O atoms with an isotopic enrichment of 31 ± 2%.
This value is significantly lower than the 18O content of
the acyl-CoA derivatives and can be explained by the partial hydrolysis
of the enzyme-coenzyme A thiol ester intermediate in
H216O. Control experiments, in which either the
glutaryl-CoA or [18O2]acetate was omitted,
showed within the experimental error no incorporation of
18O into the undecapeptide. In another control experiment,
the native 18O-labeled enzyme was separated from the excess
[18O2]acetate and subsequently incubated with
100 mM unlabeled acetate and 1 mM glutaryl-CoA.
Again no 18O was found in the undecapeptide (Table
I).
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In addition to the peptide signals at 1274 Da, varying amounts of an oxidized form of the peptide at 1290.8 ± 1.2 Da were found. The mass difference of 16 Da suggested an oxidation of the methionine 58 to the sulfoxide. Further analysis showed that this oxidation was inevitable during the preparation of peptide samples for MALDI-TOF MS. Using a carboxyl-terminal cleavage with carboxypeptidase A and MALDI-TOF MS read-out of the peptide ladder, the C-terminal sequence VESGLM of the peptide was confirmed. The oxidation site was located on methionine 58 and the 18O label of the peptide was found in glutamate 54 (data not shown). The 18O contents in the non-oxidized and the oxidized forms of the peptide were found to be identical within the experimental accuracy. However, the isotopic distribution of the oxidized form of the peptide was always slightly disturbed by the sodium adduct of the non-oxidized peptide.
As evident from Fig. 2, wild-type glutaconate CoA-transferase also catalyzed the slow hydrolysis of glutaryl-CoA. Further analysis revealed that this hydrolytic activity was significantly stimulated by 100 mM acetate (114 milliunits/mg as compared with <2 milliunits/mg, respectively), whereas the hydrolysis of acetyl-CoA was not further enhanced (105 milliunits/mg as compared with 98 milliunits/mg). Propionyl-CoA was hydrolyzed more slowly than acetyl-CoA, and a stimulation by 100 mM acetate was found (54 and 118 milliunits/mg, respectively) (Table II). It should be noted that the apparent Km values observed for the hydrolysis of different acyl-CoA derivatives were about 500 times lower (67-69 µM, Table II) than the apparent Km value for acetate (26 mM) in the CoA-transferase reaction in the presence of 100 µM glutaryl-CoA (4).
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In this paper we have demonstrated that a mechanism-based introduction
of 18O label can be followed during several steps of
analysis to the level of the labeled amino acid and that quantitative
results can be obtained from the integrated spectra. These results
encouraged us to investigate the hydrolysis reaction catalyzed by
wild-type glutaconate CoA-transferase. As expected, the chemical
hydrolysis of the HPLC-purified thiol ester between
Glu-54 and CoA in H218O at pH
8.6 led to the incorporation of almost exactly 1.0 18O into
the catalytic glutamate residue (47 ± 3%, Table I). By enzymatic
hydrolysis of acetyl-CoA in 50% H218O
catalyzed by wild-type glutaconate CoA-transferase, both the remaining
acetyl-CoA (0.85 mM) and the Glu-54 residue
became equally labeled (14 ± 3% and 12 ± 3%, respectively
(Table I).
A glutaconate CoA-transferase, in which the catalytic glutamate
has been replaced by aspartate by site-directed mutagenesis, has been
shown to be a thiol ester hydrolyase rather than a coenzyme A-transferase. Although the replacement of glutamate by aspartate is
predicted to generate an additional cleavage site for endoproteinase Asp-N, this new cleavage site was apparently not used under
the experimental conditions. If this enzyme was incubated in the
presence of either [18O2]acetate and
glutaryl-CoA or glutaryl-CoA in H218O, no
18O was found in the corresponding undecapeptide even after
complete hydrolysis of glutaryl-CoA. In addition, the formation of an
enzyme-CoA thiol ester was never observed with this enzyme.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The incubation of [18O2]acetate and glutaryl-CoA in the presence of glutaconate CoA-transferase from A. fermentans redistributed the 18O label, initially present only in acetate, among all seven of the participating oxygen atoms. Whereas the 18O content of the three oxygen atoms of glutaryl-CoA increased immediately, the decrease in the 18O content of the single oxygen atom of acetyl-CoA was slightly delayed. This observation is in agreement with the statistical proposal for the exchange reaction. Initially, the reaction proceeds predominantly in the direction of acetyl-CoA formation. Hence, the initially formed acetyl-CoA is predicted to carry the same 18O content as the added [18O2]acetate. Moreover, after a few catalytic cycles, the oxygen atoms of the catalytic glutamate had almost the same 18O content as the [18O2]acetate. Hence, all glutarate molecules released from glutaryl-CoA are 18O-labeled, and the reformed glutaryl-CoA contained 18O.
The data demonstrate the exchange of oxygen between
[18O2]acetate and glutaconate CoA-transferase
as predicted by the formation of a mixed anhydride between actetate and
the catalytic glutamate residue during catalysis of CoA-transfer. The
observed exchange was found to require the presence of both
[18O2]acetate and glutaryl-CoA and was shown
to be completely reversible (Table I). The 18O label has
been found to be localized on both oxygen atoms of glutamate residue 54 within the -subunit of the enzyme, which forms the enzyme-CoA thiol
ester intermediate as predicted by the mechanism outlined above (Fig.
1). Only one oxygen atom of the catalytic glutamate residue became
labeled with 18O by chemical hydrolysis of the isolated
thiol ester between CoA and the wild-type enzyme in
H218O.
For the E54D mutant of glutaconate CoA-transferase, it
was proposed that because of the shortened side chain of aspartate as
compared with the original glutamate residue, a water molecule might
occupy the space between the aspartate and the acyl-CoA in the active
center (9). Acting as a general base, the carboxylate group of
aspartate has been suggested to activate the water for hydrolysis of
the thiol ester, which is in agreement with the absence of any
18O in the Asp-54 carboxylate. This result,
however, cannot exclude the intermediacy of a mixed anhydride between
Asp-54 and acetate, which is specifically attacked by
H218O at the acetyl carbonyl (Fig.
6). Interestingly, the slow hydrolysis of
acetyl-CoA in H218O catalyzed by the wild-type
enzyme led to a significant 18O incorporation into the
Glu-54 carboxylate. In this case at least three mechanisms are
possible that cannot be distinguished because of the subsequent much
faster equilibration of the four oxygen atoms of acetate and Glu-54
by CoA-transfer. The three mechanisms are as follows: (i) hydrolysis of
acetyl-CoA by assistance of Glu-54 carboxylate; (ii) hydrolysis of
either carbonyl of the mixed anhydride; or most likely, (iii) the
acetate catalyzed hydrolysis of the enzyme-thiol ester.
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Although the low but significant CoA-ester hydrolyase activity has been
recognized much earlier (4), this current work shows that hydrolysis is
due to an intrinsic property of the enzyme and subject to catalysis by
acetate. Whereas glutaryl-CoA was not hydrolyzed (<2 milliunits/mg),
hydrolyase activity was observed (114 milliunits/mg) in the presence of
100 mM acetate exhibiting an apparent Km = 69 µM acetate (Table II). Further analysis revealed
that acetyl-CoA is hydrolyzed at the same rate and is not affected by
the addition of acetate, whereas propionyl-CoA behaves in a manner in
between acetyl- and glutaryl-CoA. The low apparent
Km for acetate observed in the hydrolysis of acyl-CoA substrates is comparable with the low apparent
Km-values observed in CoA-ester hydrolysis by the
E54D mutant and for the specific acetyl-CoA-dependent
exchange of 1.0 3H from [2,4-3H]glutaconate
with the solvent water (14). These low Km values
show that the enzyme is able to bind these substrates very tightly. In
all three reactions no complete CoA-transfer occurred. The unusually
high Km-values in the complete CoA-transfer are in
accordance with the hypothesis of White and Jencks (7) that
CoA-transferases are able to convert binding energy into rate
enhancement. If one adds the ~370-fold increase in
Km for acetate and the ~170-fold increase in
Km for acetyl-CoA (14), the transition state could
be lowered by 27 kJ/mol.
It has been shown by reduction with NaBH4 (4) and by
MALDI-TOF MS (this paper) that incubation of the CoA-transferase with glutaryl-CoA alone results in up to 100% conversion of the enzyme into
the stable CoA-ester. Addition of acetate results in two reactions,
CoA-transfer and hydrolysis. If hydrolysis requires a proceeding
CoA-transfer, then no difference should be observed in the
Km for both processes. Hence, it appears likely that
acetate stimulates the hydrolysis of the enzyme CoA-ester intermediate
by activating a water molecule. One of the possible two carboxylate
binding sites should be large enough to accommodate water together with
the general base acetate or, to a lesser extent, with propionate (Fig.
7). The hydrolysis at the stage of the
enzyme-CoA ester rather than acyl-CoA is in accordance with the high
reactivity of the former toward NaBH4. Probably the
enzyme-CoA ester is somehow distorted, which makes the carbonyl group
more accessible for nucleophiles.
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In E. coli 3-methyladenine-DNA glycosylase II (DNA
glycosidase, AlkA) the catalytic aspartate residue 238 of the enzyme
was proposed to activate a water molecule for hydrolysis of the
N-glycosidic bond of the substrate (15). In agreement with
this mechanism, it was found that the exchange D238N by mutagenesis
completely abolished the hydrolyase activity of the protein. The same
holds true for the mutants of glutaconate CoA-transferase (9, 10). Whereas the E54Q mutant is slowly converted to the active wild-type CoA-transferase in the presence of both substrates, no reactivation to
a hydrolyase was found for the E54N mutant. In a mechanism-based reactivation of the E54Q mutant, the substrate carboxylate,
e.g. glutaconate, may slowly form a mixed anhydride with the
glutamine residue, and ammonia is formed. The anhydride is further
hydrolyzed or more likely aminolyzed, yielding the active, glutamate
54-containing enzyme. In contrast, the E54N mutant is not
converted to the hydrolyase in a similar, mechanism-based fashion,
probably because the anhydride between the aspartate and the
glutaconate residue cannot be formed due to a gap between the
asparagine 54 and the enzyme-bound dicarboxylate.
The following lines of evidence support the activation of a water
molecule for hydrolysis of acyl-CoA by the E54D mutant of
glutaconate CoA-transferase rather than formation of a mixed anhydride
intermediate: 1) no 18O incorporation from
H218O or
[18O2]acetate was found for the E54D
mutant enzyme in the presence or absence of acyl-CoA; 2) no
reactivation to a hydrolyase activity was found for the E54N mutant,
whereas the E54Q mutant could be reactivated to the active wild-type
enzyme in the presence of substrates; and 3) a specific acyl-CoA
hydrolyase activity with acetate as cofactor was found for the
wild-type enzyme.
Taking all of these findings together, it appears far more likely that
the carboxyl group of aspartate can act as a general base in the
activation of a water molecule, allowing the direct hydrolysis of the
acyl-CoA substrate rather than the intermediate formation of a mixed
anhydride, which is further hydrolyzed.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Prof. R. Thauer, Max-Planck-Institut fuer Terrestrische Mikrobiologie (Marburg, Germany) for permission to use his MALDI-TOF mass spectrometer and A. Willanzheimer for technical support. T. S. acknowledges fruitful and stimulating discussion with Dr. A. Pierik, Laboratorium für Mikrobiologie, Philipps University, Marburg.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.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.
To whom correspondence should be addressed. Fax: 049-6421-288979;
E-mail: selmer@mailer.uni-marburg.de.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; HPLC, high pressure liquid chromatography.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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