(Received for publication, October 13, 1995; and in revised form, December 21, 1995)
From the
In methanogens, the acetyl-CoA decarbonylase synthase (ACDS)
complex, which has five different subunits, catalyzes synthesis and
cleavage of acetyl-CoA according to the reaction: CO +
2H
+ 2e
+
CH
-H
SPt + CoA &lrhar2; acetyl-CoA +
H
SPt + H
O, where H
SPt and
CH
-H
SPt are tetrahydrosarcinapterin and N
-methyl-tetrahydrosarcinapterin,
respectively. We have dissociated the ACDS complex into three protein
components by limited proteolytic digestion. Catalysis of acetyl-CoA
synthesis was lost in parallel with the loss of the intact
subunit; however, no decrease in activity was detected in any of three
partial reactions found to be catalyzed by distinct protein components
of the proteolyzed ACDS complex: (a) CO dehydrogenase,
catalyzed by the
component, (b)
CH
-H
pteridine:cob(I)amide-protein
methyltransferase, catalyzed by the intact
subunit and fragments
of the
subunit, and (c) acetyltransferase, catalyzed by
a truncated form of the
subunit. The results indicated that the
subunit is responsible for binding CoA and acetyl-CoA and
suggested that acetyl-enzyme formation occurs on the
subunit. A
value of 5.5
[H
]
M
was determined for the equilibrium
constant of the following reaction at pH 7.5 and 25 °C:
CH
-H
SPt + cob(I)amide-protein +
H
&lrhar2; H
SPt +
CH
-cob(III)amide-protein.
The acetyl-CoA decarbonylase synthase (ACDS) ()complex has been detected in a variety of methanogens
including species of Methanosarcina, Methanothrix (i.e. Methanosaeta), and Methanococcus(1, 2) . (
)(
)The multienzyme complex from Methanosarcina barkeri is composed of five different subunits,
possibly arranged in an
structure with the individual subunits of molecular masses of 89,
60, 50, 48, and 20 kDa, respectively(3) . The complex contains
CO:acceptor oxidoreductase, Co-
-methylcobamide:tetrahydropteridine
methyltransferase, and acetyl-CoA synthase
activities(3, 4, 5) . In the past, the ACDS
complex has been referred to as the carbon monoxide dehydrogenase
complex and/or the carbon monoxide dehydrogenase-corrinoid enzyme
complex. Previous investigations of acetyl-CoA synthesis and cleavage (3, 5) have established that the purified, intact
enzyme complex catalyzes the following reaction,
where Fd stands for ferredoxin, and
CH-H
SPt and H
SPt denote N
-methyl-tetrahydrosarcinapterin and
tetrahydrosarcinapterin, respectively. The sarcinapterin compounds are
used in M. barkeri in place of the corresponding
tetrahydrofolate derivatives. The structures of
CH
-H
SPt and H
SPt are shown in Fig. 1.
Figure 1:
Structures of tetrahydrosarcinapterin
(HSPt), R = H; and N
-methyl-tetrahydrosarcinapterin
(CH
-H
SPt), R = CH
(20) .
The overall reaction of acetyl-CoA synthesis or cleavage may be divided into several possible partial reactions. One of these is CO:acceptor oxidoreductase (CO dehydrogenase). This reaction may be written as follows,
and is carried out by an component, containing nickel and iron-sulfur centers, as shown in
studies (6) that preceded those on the multienzyme complex.
This partial reaction is catalyzed also by the intact ACDS complex from
the genus Methanosarcina(1, 3) , from Methanothrix strain CALS 1,
and from Methanococcus vannielii, (
)as well as by the
component isolated from the
multienzyme complexes from M. barkeri(2, 6) , M. vannielii(7) , and Methanosarcina
thermophila(8) . The
protein in Methanothrix sohengenii has been reported to
exhibit an oxygen-stable CO dehydrogenase activity and has also been
the subject of several detailed
studies(9, 10, 11, 12) .
Interestingly, evidence for a high molecular mass enzyme complex in
this organism has not yet been presented.
A discrete, 102 kDa,
corrinoid/iron-sulfur protein
subcomponent of the ACDS complex from M. thermophila has been
identified in experiments in which the complex was dissociated by
treatment with a cationic detergent(8) . In these studies, a
portion of the enzyme complex remained undissociated, and recovery of
the
subunit was not reported. It was shown that the reduced
corrinoid protein became methylated in the presence of methyl iodide.
However, methylation by CH
-H
SPt was not
described, and it was unknown whether or not a separate
methyltransferase enzyme, analogous to that required for methylation of
the clostridial 88-kDa corrinoid/iron-sulfur protein by N
-methyl-tetrahydrofolate (13, 14) was involved in methyl group transfer from
CH
-H
SPt.
The purpose of the present
investigation was to investigate fundamental structural and functional
properties of subunit/subprotein interactions in studies on the
quaternary structure of the ACDS enzyme complex. The results include
development of a method for quantitative dissociation of the enzyme
complex along with the isolation and characterization of a truncated
form of one of the subunits and two other protein components with
characteristic subunit compositions. A new, nonradioactive method was
developed for analysis of acetyltransferase activity. Identification
was made of the protein subcomponents involved in catalysis of two
additional partial reactions, that of
Co--methylcob(III)amide:tetrahydropteridine methyltransferase (), and acetyltransferase ().
A strong correlation was shown between the ability of the ACDS
complex to catalyze and the level of intact 60-kDa
subunit that remains during proteolytic digestion. Finally, experiments
are described in which determination is made of the K`
of . The significance of the value of the K`
to methyl group transfer in vivo is
discussed.
Prior to assay for acetyltransferase activity, samples
from the fractions were reduced by incubation at one-tenth of their
original concentration for 10 min in the presence of 2 mM Ti-EDTA in 50 mM MOPS buffer, pH 7.2,
in a total volume of 100 µl. Aliquots (5-10 µl) of the
incubation mixtures were then added to a solution (97-92 µl)
containing 18 nmol of acetyl-CoA, 0.8 nmol of ferredoxin, 460 nmol of
Ti
-EDTA, and 6 µmol of MOPS buffer, pH 7.2. The
acetyl transfer reaction was initiated by the addition of 18 µl of
1 mM 3`-dephosphocoenzyme A. The reaction mixtures (final
volume of 120 µl) were maintained at 23 °C for 20 min.
Thereafter, the reactions were stopped by addition of 120 µl of a
solution containing 20 mM sodium 2-mercaptoethanesulfonate and
0.5 M sodium citrate, pH 4.0. The final mixtures were stored
frozen in liquid nitrogen prior to analysis. Quantitative determination
of the products coenzyme A and S-acetyl-3`-dephospho-CoA was
carried out on samples, 100 µl, analyzed by reversed phase HPLC
under anaerobic conditions. HPLC analysis was carried out as described
previously (3, 5) with modification of the solvent
gradient to allow separation of the four derivatives: coenzyme A,
3`-dephospho-CoA, acetyl-CoA, and S-acetyl-3`-dephospho-CoA.
In the modified procedure, the column (APEX octadecyl, 250
4.6
mm diameter, from Jones Chromatography, Inc.) was equilibrated in 50
mM tetramethylammonium phosphate, pH 4.7, and a linear
gradient of 0-20% acetonitrile in the same solution was applied
at 1 ml/min over a period of 35 min. As shown in Fig. 2, the
rate of product formation declines steadily as the reaction proceeds.
Therefore, the reaction time and amount of enzyme employed for single
time point assays of fractions was chosen so that none of the reactions
were allowed to proceed to more than approximately 30% completion. The
rate of acetyl-CoA hydrolysis was found to be negligible in control
reactions carried out in the absence of 3`-dephospho-CoA. Formation of S-acetyl-3`-dephospho-CoA was not detected in reaction
mixtures that lacked Ti
-EDTA.
Figure 2:
Acetyl transfer reaction catalyzed by the M. barkeri ACDS complex. Analysis of the reaction of acetyl
group transfer from acetyl-CoA to 3`-dephospho-CoA catalyzed by the
intact ACDS complex was carried out by the method described for assay
of acetyltransferase activity in resolved fractions of the proteolyzed
enzyme, with the following modifications. Preincubation of the enzyme
complex (57 µg) was carried out at room temperature (27
°C) for 1.5 min under strictly anaerobic conditions in a mixture
(50 µl total volume) containing approximately 0.8 nmol of
ferredoxin, 0.2 µmol Ti
-EDTA and 50 mM MOPS buffer, pH 7.2. An aliquot (15 µl) of the incubation
mixture was then transferred to a solution (550 µl), which
contained, except for dephospho-CoA, all other components of the
reaction (90 nmol of acetyl-CoA, 3.2 µmol of
Ti
-EDTA, 0.8 nmol of ferredoxin, and 30 µmol of
MOPS-Na, pH 7.2). This mixture was incubated for an additional 1.5 min,
and the reaction (final volume 600 µl) was initiated by the
addition of 3`-dephospho-CoA (35 µl of a 2.58 mM stock
solution). The reaction was allowed to proceed at 27 °C. Samples
(60 µl) were removed periodically, mixed with an equal volume of 20
mM sodium 2-mercaptoethanesulfonate, 0.5 M sodium
citrate, pH 4.0, to stop the reaction, and frozen for subsequent HPLC
analysis. Based on the initial rate of the reaction, the specific
activity of acetyltransferase was found to be 1.2 µmol of acetyl
transferred per min/mg of enzyme complex.
The ability of limited proteolytic digestion to cause
dissociation of the M. barkeri ACDS complex was investigated
by reaction of the enzyme complex with chymotrypsin followed by
anion-exchange chromatography. As shown in Fig. 3, this
procedure resulted in resolution of three major peaks of protein. The
three protein components emerged from the column following a peak of
unbound material that contained peptides too small to be resolved on a
12% acrylamide SDS gel. Each of the three protein peaks possessed
characteristic subunit compositions, as shown by SDS-PAGE analysis (Fig. 3). Unaltered subunit bands as well as modified subunits
were identified by Western blot analyses using subunit-specific
antibodies (data not shown). As shown in Fig. 3, the first
protein peak contained the subunit and four other polypeptides
corresponding to partially degraded forms of the
subunit
(
*). The second protein component contained the
and
subunits. The final peak contained a truncated form of the
subunit (
*). Evidence for the presence of iron-sulfur centers in
each of the three peaks was found both by direct determinations of iron
and by measurement of the UV-visible absorption spectra of the three
protein components. No fraction was recovered that contained detectable
quantities of the unresolved enzyme complex. Therefore, the overall
dissociation of the complex was judged to be efficient and
quantitative.
Figure 3:
Resolution of the ACDS complex from M.
barkeri into individual protein subcomponents. Subcomponent
proteins of the enzyme complex were isolated by limited digestion of
the enzyme complex with chymotrypsin followed by anion-exchange
chromatography, as described under ``Materials and Methods.''
Chromatography was monitored based on absorbance at 280 nm. Samples of
the undigested enzyme and each of the three protein peaks shown were
analyzed by SDS gel electrophoresis (see inset). All of the
subunits indicated were identified by use of subunit-specific
antibodies in Western blot analyses (not shown). Peak 1 contained the
subunit and four other polypeptides corresponding to partially
degraded forms of the
subunit (
*). Peak 2 contained the
and
subunits. Peak 3 contained a truncated form of the
subunit (
*) and a lower intensity band likely to represent a
contaminant. (Samples containing the truncated
subunit have been
obtained that possess high acetyltransferase activity and are free from
additional bands. Thus, activity due to the minor band in the lanes
marked undigested and peak 3, is ruled out.) Material not bound to the
column (FLOW THROUGH) contained peptides too small to be
resolved on the 12% polyacrylamide gel (not
shown).
The extent of proteolytic digestion was varied by
incubating samples of the enzyme complex at a fixed concentration with
different concentrations of chymotrypsin. SDS-PAGE was used to monitor
the progress of digestion of each of the subunits. The band
corresponding to the subunit was found to be highly susceptible
to proteolytic attack (Fig. 4). As the concentration of
chymotrypsin was increased, the
subunit band intensity decreased
markedly. Complete loss of the intact
subunit band occurred under
conditions in which intense bands were still found for all other
subunits. Densitometric analyses showed that loss of the
band
occurred with concomitant loss of the ability to carry out overall
synthesis of acetyl-CoA, as shown in Fig. 4. Further digestion
at the levels greater than those shown in Fig. 4then resulted
in selective loss of the band corresponding to the intact
subunit. Samples that contained residual amounts of the intact
band were found to be only partially resolved by subsequent
ion-exchange chromatography. These findings indicated that activity is
lost prior to extensive dissociation of the complex.
Figure 4:
Chymotrypsin digestion of the ACDS
complex, correlation of acetyl-CoA synthesis activity with the presence
of various subunits detected by SDS-PAGE. Digestion of the enzyme
complex (5 mg/ml) with chymotrypsin was carried out by a procedure
similar to that described under ``Materials and Methods,''
with the exception that the reaction time was decreased to 20 min. The
weight ratio of chymotrypsin to enzyme complex protein is indicated and
was varied over a range of 1:3200-1:400. Incubations were
performed sequentially, and at the end of each incubation time period,
an aliquot was removed and immediately assayed for acetyl-CoA synthesis
using the substrates CH-H
SPt, CoA, and CO
(as bicarbonate), in the presence of ferredoxin and
Ti
-EDTA according to the method described
previously(5) . At the same time, the remaining digestion
mixture was mixed with an equal volume of 2
concentrated SDS
sample buffer (16) and kept for analysis by SDS-PAGE. The rate
of acetyl-CoA synthesis observed in the absence of chymotrypsin was
taken as 100% activity. Band area (%) corresponds to the
densitometrically determined areas of peaks at the positions of the
indicated subunits expressed as a percentage of the areas detected for
the same subunits in the undigested enzyme
complex.
Although
overall acetyl-CoA synthesis activity was abolished by proteolytic
treatment of the enzyme complex, no decrease in activity was detected
for CO dehydrogenase,
CH-H
pteridine:cob(I)amide-protein
methyltransferase, or acetyltransferase. Assays for total cobamide
content were carried out on fractions obtained in resolution of the
protein subcomponents, as shown in Fig. 5A. The major peak
of cobamide was closely associated with the first protein peak, as
shown in Fig. 5A. Peak 1 contained 74% of the total
cobamide. The remaining 26% eluted over a broad region of the gradient
and did not coincide with any of the three major protein peaks.
Figure 5: Analysis of cobamide content, CO dehydrogenase, and acetyltransferase activity of resolved ACDS protein subcomponents. The activities of CO dehydrogenase and acetyltransferase, and the concentration of cobamide was measured in fractions obtained following limited proteolytic digestion and anion-exchange chromatographic resolution of the ACDS subcomponent proteins, as described under ``Materials and Methods.'' Panel A, the fractions were assayed for cobamide concentration by a method that employed differential spectrophotometric detection of the reaction with cyanide, as described previously(3) . Panel B, CO dehydrogenase activity was measured by CO-dependent reduction of methylviologen as described previously(2) . Panel C, individual fractions were assayed for acetyltransferase activity, as described under ``Materials and Methods.'' Activity is expressed as nmol of S-acetyl-3`-dephospho-CoA formed per min/ml of fraction.
Since Co--methylcob(III)amide:tetrahydropteridine
transmethylation had not been reported with isolated components of the
ACDS complex, tests were performed to determine whether or not the
reduced peak 1 corrinoid protein was capable of undergoing direct
reaction with CH
-H
SPt. The Co(I) form of the
corrinoid protein was first generated by reaction with 1 mM Ti
-EDTA. As described previously for detection
of the reduced cobamide in the intact complex(4) , reduction to
the Co(I) level was indicated by the development of absorption spectra
that showed a prominent peak at 394 nm. The reduction reached 50%
completion in approximately 35 s, as shown in Fig. 6. In
contrast, under otherwise similar conditions, the undigested enzyme
complex required tens of minutes to become reduced(5) .
Addition of CH
-H
SPt to the reduced corrinoid
protein resulted in immediate loss of the 394-nm absorbance peak, and
concomitant increase in absorbance around 456 nm. The spectral changes
were characteristic of formation of the base-off
Co
-methyl cobamide, as observed previously in the
intact complex(4) . This finding indicated that methyl group
transfer from CH
-H
SPt does not require
additional subunit proteins (
,
, or
) and that
methyltransferase activity is an intrinsic property of the
corrinoid protein subcomponent.
Figure 6:
Reduction of the protein-bound cobamide
with Ti-EDTA and reaction with
CH
-H
SPt. Reduction of the ACDS corrinoid
protein subcomponent and titration with CH
-H
SPt
was carried out at 23 °C with a solution (800 µl) that
contained 50 mM Tris
HCl, pH 7.5, and the corrinoid
protein (3.55 µM cobamide) obtained from ion-exchange
chromatography of the proteolytically digested enzyme complex (peak 1
in Fig. 3). The processes of reduction by Ti
and methylation by CH
-H
SPt were monitored
spectrophotometrically. At the indicated time point 12 µl of 64
mM Ti
-EDTA was added. Addition of the
indicated amounts of CH
-H
SPt was made from a
0.485 mM stock solution. After each addition of
CH
-H
SPt, two 60-µl samples were removed
from the cuvette, mixed with 60 µl each of a solution containing 20
mM 2-thioethanesulfonate and 0.5 M sodium citrate, pH
4.0, and frozen in liquid nitrogen. In each sample, the
[H
SPt]/[CH
-H
SPt]
concentration ratio was subsequently determined by HPLC analysis
according to the procedure described previously(5) . Absorbance
data have been corrected for dilution resulting from reagent
additions.
CO dehydrogenase was also measured
in the fractions derived from anion-exchange chromatography of the
digested enzyme complex. As shown in Fig. 5B, CO
dehydrogenase was associated exclusively with peak 2. These results
constitute the first demonstration that the carbon monoxide
dehydrogenase subcomponent may be released from the enzyme complex by
the action of a protease.
Acetyltransferase activity of the intact
complex from M. thermophila was previously detected based on
the exchange of 3`-[P]CoA with
acetyl-CoA(19) . However, the subunit location of this activity
was not investigated. In order to identify the protein component
responsible for acetyltransferase activity, fractions obtained during
resolution of the digested enzyme complex were assayed for
acetyltransferase (), as described under ``Materials
and Methods.'' As shown in Fig. 5C,
acetyltransferase activity was found to be associated with the protein
peak containing the truncated
subunit. The results establish a
previously unrecognized activity of the
subunit and indicate that
the binding site for CoA and acetyl-CoA reside within a domain that
remains intact in the truncated
subunit.
Titration of the
reduced corrinoid protein with CH-H
SPt was
carried out in order to determine the equilibrium constant for methyl
transfer in . As shown in Fig. 6, each successive
addition of CH
-H
SPt made to the reduced
corrinoid protein resulted in an immediate decrease in the absorbance
function (A
-A
). The addition of excess H
SPt to the methylated
corrinoid protein caused an immediate increase in the absorbance
function, indicating that the reaction was freely reversible. The ratio
of methylated to demethylated corrinoid protein,
[CH
-cob(III)amide-protein]/[cob(I)amide-protein],
was determined from the spectrophotometric data. Corresponding ratios
of
[H
SPt]/[CH
-H
SPt]
were measured by HPLC analysis (5) of aliquots that were
removed after each addition of CH
-H
SPt, as
described in the legend to Fig. 6. Both sets of ratios are
plotted in Fig. 7as a function of the total concentration of
CH
-H
SPt added. The equilibrium
product/substrate ratio,
[H
SPt][CH
-cob(III)amide]/[CH
-H
SPt][cob(I)amide)],
was found to be independent of the amount of
CH
-H
SPt added, as shown in Fig. 7. The K`
value measured at pH 7.5 was 5.5 ± 0.3.
Figure 7:
Determination of the equilibrium constant K` for the
CH
-H
SPt:cob(I)amide-protein methyl transfer
reaction (). The K`
for the
CH
-H
SPt:cob(I)amide methyltransferase reaction
was determined by analysis of the
[CH
-cob(III)amide]/[cob(I)amide] and
[H
SPt]/[CH
-H
SPt]
concentration ratios that resulted during titration of the reduced
corrinoid protein with CH
-H
SPt as described
under ``Materials and Methods,'' and in the legend to Fig. 6. The
[CH
-cob(III)amide]/[cob(I)amide]
concentration ratios (
) were obtained from the spectrophotometric
data, and the corresponding
[H
SPt]/[CH
-H
SPt]
concentration ratios (
) were determined by HPLC analysis, as
indicated in the legend to Fig. 6. The concentration ratios, and
the corresponding K`
values (
) obtained as
the product
[CH
-cob(III)amide]/[cob(I)amide]
[H
SPt]/[CH
-H
SPt]
are shown plotted as a function of the total added
CH
-H
SPt.
It was demonstrated previously that the ACDS complex catalyzes the synthesis and cleavage of acetyl-CoA ()(3, 5) ; however, unambiguous assignment of the catalytic roles of the various protein subcomponents of the enzyme complex has not been reported. In order to obtain information on the quaternary structure of the ACDS complex and to identify catalytic properties of individual protein subunits or subcomponents, we developed a new procedure for dissociation of the enzyme complex. Important advantages over the previously described detergent fractionation protocol (8) are (a) that three protein components are resolved instead of two and (b) that dissociation of the enzyme complex is quantitative and does not produce a fraction corresponding to the residual unresolved enzyme complex. The three separate protein components so obtained display distinct subunit compositions and exhibit characteristic catalytic activities.
Equilibrium studies of methyl transfer between the enzyme-bound
corrinoid moiety and the reduced pteridine substrate indicate that the
process is freely reversible under physiologically relevant conditions.
The equilibrium constant for
CH-H
SPt:cob(I)amide-protein methyl transfer () was 5.5 [H
]
M
(Fig. 7). Therefore, in the
process of acetyl-CoA cleavage, methyl group transfer from the enzyme
to H
SPt is slightly thermodynamically unfavorable under
standard state conditions (K`
= 1/5.5,
G
` = +1.0 kcal/mol). However,
very low levels of CH
-H
SPt are detected during
purification of H
SPt from cell extracts, and based on the
amounts of ACDS complex and H
SPt obtained during
purification from an equal amount of cell paste, the ratio of
H
SPt to enzyme cobamide is estimated to be approximately
50:1. These findings indicate that at equilibrium in the presence of
physiological concentrations of enzyme and
CH
-H
SPt, approximately 91% of the enzyme-bound
methyl groups would be transferred to the cellular pool of
H
SPt. Demethylation of the enzyme corrinoid is also
exceedingly rapid. Therefore, it is unlikely that methyl group transfer
to H
SPt presents either a kinetic or thermodynamic barrier
of significance in the overall process of acetyl-CoA cleavage in
vivo.
In summary,
the results demonstrate catalytic roles for the (or possibly
alone),
, and
protein subcomponents of the ACDS
complex. We may now write reactions 5-7 to include the enzyme
subcomponents with a subscript denoting the relevant subunits.
The subunit catalyzes the exchange/transfer of the acetyl
group of acetyl-CoA. Consequently a likely role for the
subunit
in acetyl-CoA synthesis in the intact ACDS complex would be to catalyze
the reversible synthesis of acetyl-CoA from CoA and an acetyl group
bound to the
subunit. The role of the
subunit in the intact
ACDS complex would be to catalyze the reversible transfer of the methyl
group from CH
-H
SPt to the bound cobamide
cofactor, and perhaps ultimately to the site of acetyl synthesis. The
exact role of the
subunit is unknown, however, our data suggest
that integrity of the
subunit could be involved in maintaining
the overall quaternary structure. During the process of acetyl-CoA
cleavage, the transfer of the methyl group from the corrinoid protein
to the cellular pool of H
SPt is rapid and thermodynamically
favorable under conditions likely to exist in vivo.