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J Bacteriol, March 1998, p. 1129-1134, Vol. 180, No. 5
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Essential Glutamates in the
Acetate Kinase from Methanosarcina thermophila
Kavita
Singh-Wissmann,1,2,3
Cheryl
Ingram-Smith,1
Rebecca D.
Miles,1 and
James G.
Ferry1,2,*
Department of Biochemistry and Molecular
Biology, The Pennsylvania State University, University Park,
Pennsylvania 16802-45001;
Department of
Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute
and State University, Blacksburg, Virginia
240612; and
Department of Molecular
Microbiology, Washington University School of Medicine, St. Louis,
Missouri 631103
Received 9 October 1997/Accepted 10 December 1997
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ABSTRACT |
Acetate kinase catalyzes the reversible phosphorylation of acetate
(CH3COO
+ ATP
CH3CO2PO32 + ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of
the enzyme from Escherichia coli indicate an unspecified
glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273-280, 1974). Alignment
of the amino acid sequences for the acetate kinases from E. coli (Bacteria domain), Methanosarcina
thermophila (Archaea domain), and four other
phylogenetically divergent microbes revealed high identity which
included five glutamates. These glutamates were replaced in the
M. thermophila enzyme to determine if any are essential for
catalysis. The histidine-tagged altered enzymes were produced in
E. coli and purified to electrophoretic homogeneity by
metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is
essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the Km values
for acetate and ATP with only moderate decreases in
kcat, which suggests that this residue is
involved in substrate binding but not catalysis. The unaltered acetate
kinase was not inactivated by N-ethylmaleimide; however,
replacement of E385 with cysteine conferred sensitivity to
N-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located
near the active site. Replacement of E97 decreased the Km value for acetate but not ATP, suggesting
this residue is involved in binding acetate. Replacement of either E32
or E334 had no significant effects on the kinetic constants, which
indicates that neither residue is essential for catalysis or
significantly influences the binding of acetate or ATP.
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INTRODUCTION |
Acetate is an end product of most
fermentative microbes and is the major growth substrate for the
methanoarchaea (22); thus, carbon flow through acetate is of
primary importance in anaerobic microbial consortia and the global
carbon cycle. Although the metabolisms of fermentatives and
acetotrophic methanoarchaea represent the extremes of biochemical
divergence in energy-yielding pathways, these microbes have in common
the enzymes acetate kinase (reaction 1) and phosphotransacetylase
(reaction 2).
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(1)
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(2)
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These two enzymes produce acetate from acetyl coenzyme A
(acetyl-CoA) in the fermentatives, where a major portion of the energy
requirements is obtained through substrate-level phosphorylation catalyzed by acetate kinase. The methanosarcinas utilize acetate kinase
and phosphotransacetylase to activate acetate to acetyl-CoA in the
first step of the pathway for the fermentation of acetate to methane
(5). The acetyl-CoA is cleaved by the CO
dehydrogenase-acetyl-CoA synthase enzyme complex, yielding methyl and
carbonyl groups (5). The methyl group is reduced to
CH4 with electrons derived from oxidation of the carbonyl
group to CO2. Transport of electrons through a
membrane-bound transport chain generates an electrochemical ion
gradient driving ATP synthesis. Thus, acetate kinase and
phosphotransacetylase are at the interface of energy-yielding
metabolism between fermentatives and the acetotrophic methanoarchaea,
which are the principal metabolic groups in anaerobic consortia
degrading complex organic matter to methane. In addition to a key
intermediate in energy metabolism, acetylphosphate acts as a phosphoryl
donor to enzyme I of the phosphoenolpyruvate:glucose phosphotransferase
system in Escherichia coli and Salmonella
typhimurium via a phosphoenzyme intermediate of acetate kinase
(7). Acetylphosphate is also a phosphoryl donor to
periplasmic binding proteins (8) as well as to many response
regulator proteins of two-component systems (13). It has
been proposed that acetylphosphate functions as a global regulatory signal in E. coli (13, 20). Thus, acetate kinase
influences the physiology of diverse microbes in a variety of ways.
Comparison of deduced amino acid sequences reveals high identity among
acetate kinases (Fig. 1) from the
Archaea and Bacteria, which suggests that this
enzyme was either highly evolved prior to divergence of the domains or
that horizontal gene transfer occurred between domains. The high
identity suggests similar mechanisms for these acetate kinases. The
acetate kinase from E. coli is phosphorylated with
acetylphosphate or ATP (3, 6, 19), and it has been proposed
that a phosphoryl-enzyme intermediate is involved in the catalytic
mechanism. Acid hydrolysis of the phosphorylated enzyme reduced with
[3H]borohydride yields
[3H]
-amino-
-hydroxyvaleric acid, suggesting that an
unspecified 
-phosphorylated glutamyl residue is involved in
catalysis (19). This hypothesis is novel, as there are no
reports of a phosphorylated glutamate in any enzyme reaction mechanism.
The proposed mechanism was challenged when it was shown that
phosphorylated acetate kinase from E. coli is a phosphoryl
donor to enzyme I of the bacterial phosphotransferase system
(7), suggesting the possibility of an alternate function for
phosphorylated acetate kinase. Furthermore, no evidence has been
reported for a glutamate which is essential for kinase activity. Thus,
the involvement of glutamate in catalysis of kinase activity by acetate
kinases is a matter of controversy.
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FIG. 1.
Alignment of the deduced amino acid sequences of acetate
kinases. Abbreviations and GenBank accession numbers: M.t., M. thermophila (L23147); E.c., E. coli (M22956); C.a.,
Clostridium acetobutylicum (U38234); B.s., Bacillus
subtilis (L17320); H.i., Haemophilus influenzae
(L45839); M.g., Mycoplasma genitalium (L43967). Amino acids
are in the single-letter code. Dashes represent gaps introduced for
alignment. Closed circles mark the glutamate residues targeted for
replacement. Identical residues are shown in bold.
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Despite the broad importance of acetate kinase in microbial physiology,
the use of biochemical genetics to probe the catalytic site has not
been reported. Here we identify an essential glutamate in the
Methanosarcina thermophila enzyme which provides support for
the previously proposed catalytic mechanism. Two other glutamates are
implicated in substrate binding.
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MATERIALS AND METHODS |
Protein sequence analyses.
Databases were searched at the
National Center for Biotechnology Information by using the BLAST
network server (2). CLUSTAL W (18) was used for
multiple protein sequence alignment at the Human Genome Center of the
Baylor School of Medicine.
Site-directed mutagenesis.
Mutagenesis was performed
according to the manufacturer's instructions with the Muta-Gene
phagemid mutagenesis kit (Bio-Rad), which employs the
oligonucleotide-directed in vitro mutagenesis method (11).
The plasmids used are shown in Table 1.
The M. thermophila acetate kinase gene was excised from
pUC19/ack (12) by KpnI and
BamHI digestion and ligated into pTZ18U to generate pTZack. The mutations were verified by double-stranded
sequence analysis by using the dideoxy chain termination method
(15) and Sequenase version 2.0 (United States Biochemicals).
Heterologous production and purification of acetate kinase.
The unaltered and altered acetate kinase genes were subcloned into the
T7-based expression vector pET15b (Novagen) to generate the plasmids
listed in Table 1. In these plasmids, a 60-nucleotide leader sequence
with six tandem histidine codons was fused in frame to the 5' end of
the unaltered and altered ack genes. E. coli
BL21(DE3) was transformed with the expression vectors, inoculated into
50 ml of Luria-Bertani medium containing 100 µg of ampicillin per ml,
and grown at 37°C to an A600 of 0.6 to 0.9, at
which time IPTG (isopropyl-
-D-thiogalactopyranoside) was
added to a final concentration of 1 mM. After 1.5 to 2.0 h of
induction, the cells were harvested and stored at 
70°C. The
unaltered and altered acetate kinases were purified using a
Ni-nitrilotriacetic acid silica spin kit (Qiagen) according to the
manufacturer's instructions. The enzymes were eluted in buffer (pH
7.0) containing 50 mM NaH2PO4, 300 mM NaCl, and
250 mM imidazole. Protein concentrations were determined by the
Bradford method (4), using protein dye reagent (Bio-Rad) and
bovine serum albumin as the standard.
Enzyme activity assays.
Acetate kinase activity was
determined by the previously described (1) standard
hydroxamate assay, which detects the formation of acetyl phosphate from
acetate (200 mM) and ATP (10 mM). The acetate concentrations were 0.7 and 1.5 M for determination of the kinetic constants for ATP for the
E385A and E385D enzymes.
The ATP-ADP exchange assays were performed as follows. The reaction
mixture (100 µl) contained the following: triethanolamine-HCl,
50 mM,
pH 7.0; ATP, 1 mM; ADP, 1 mM; MgCl
2, 2 mM; acetate kinase,
10 µg; [
-
32P]ATP (3,000 Ci/mmol), 5 µCi. The
reactions were initiated by
the addition of enzyme, and mixtures were
incubated at 20°C. At
various times, 5 µl was withdrawn, and the
reaction was stopped
by the addition of an equal volume of 1 N HCl. A
volume of 5 µl
was applied to polyethyleneimine cellulose thin-layer
chromatography
plates (Bakerflex), which were developed ascendingly in
0.52 M
potassium phosphate (pH 3.5). The plates were air dried and
autoradiographed
to visualize the ATP and ADP spots. The ADP spots were
excised
and counted in 5 ml of Scintiverse (Fisher Scientific)
scintillation
cocktail. Initial velocities were calculated as described
previously
(
16).
Inhibition by NEM.
N-Ethylmaleimide (NEM) (final
concentration, 10 µM) was added to the unaltered acetate kinase (5 µg/ml) and to the E385C enzyme (40 µg/ml) in a final volume of 100 µl at 37°C. Aliquots (10 µl) were removed at the indicated times
and assayed for kinase activity. Substrate protection experiments were
performed by preincubating the enzyme for 5 min at 37°C with (final
concentrations) ADP (10 mM), ATP (10 mM), acetyl phosphate (10 mM), or
potassium acetate (200 mM) prior to addition of NEM.
Circular dichroism spectroscopy.
Spectra were acquired at
37°C with an Aviv circular dichroism (CD) spectrometer, model 62DS.
Samples (1 to 10 µM) of acetate kinase in 20 mM sodium phosphate (pH
7.5) containing 0.1 M NaCl were placed in a cuvette with a 1-mm path
length and data points obtained from 205 to 320 nm in 1.0-nm
increments. Five spectra were taken for each sample and averaged. The
resulting spectra were normalized for direct comparison.
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RESULTS AND DISCUSSION |
The sequences of acetate kinases deduced from the genes of widely
divergent microbes from the Archaea and Bacteria
domains are highly identical, suggesting a similar catalytic mechanism (Fig. 1); for example, the enzymes from E. coli
(Bacteria) and M. thermophila
(Archaea) have 44% identity. It has been proposed that a
phosphorylated glutamate functions in a covalent catalytic mechanism
for the E. coli enzyme (19). Five glutamates in
the M. thermophila acetate kinase were selected for
replacement to determine if any are essential for catalysis. The
glutamates selected (Fig. 1) are either highly conserved and within a
region of high conservation (E97) or 100% conserved (E32, E334, E384,
and E385) with the deduced sequences for five other acetate kinases
from extremely diverse microbes. The histidine-tagged altered enzymes were heterologously produced in E. coli and purified by
one-step metal affinity chromatography. Each was judged to be
homogeneous by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, and the subunit molecular masses were
indistinguishable from that of the unaltered histidine-tagged acetate
kinase (not shown). Native gel filtration chromatography (not shown)
indicated that the altered enzymes were dimeric, in accord with the
authentic acetate kinase purified from M. thermophila
(1). The unaltered histidine-tagged acetate kinase displayed
a kcat (Table 2)
slightly greater than the value previously determined for the authentic enzyme (1) and was most likely a result of the one-step
purification procedure yielding more active enzyme. There were no
significant differences between the unaltered (Table 2) and authentic
acetate kinases with respect to the Km values
for acetate or ATP.
Replacement of E384 and E385.
Among the five glutamates
targeted (Fig. 1), all except the enzymes altered at E384 had
substantial activity (Table 2). Activity in the standard assay was
undetectable (less than 1.0 µmol/min/mg of protein) for the E384D and
E384Q enzymes. The E384A enzyme had detectable but extremely low
specific activity (4.0 ± 2.0 µmol/min/mg of protein) in the
standard assay, which was only 0.5% of the specific activity for the
unaltered acetate kinase assayed under the same conditions. The low
activity precluded reliable determinations of kinetic constants. These
results suggest that E384 is the only essential glutamate. This
glutamate is 100% conserved with all other deduced sequences for
acetate kinases from widely divergent organisms (Fig. 1), which is
consistent with an essential function. The CD spectra of the E384Q
enzyme (Fig. 2) and the E384D enzyme (not
shown) were similar to that of the unaltered acetate kinase. The
ellipticity values at 210 nm for the E384Q and E384D enzymes were 3.6 and 1.2% less negative than that for the unaltered acetate kinase.
This result suggests no global conformational change on substitution of
E384 with Q or D, which is consistent with the high recovery on
purification of the enzymes altered at E384 and suggests that they were
dimeric in accord with the authentic enzyme purified from M. thermophila. These results, combined with the loss of kinase
activity for the enzymes altered at E384, suggest that E384 is
essential for catalysis, which is consistent with the previously
proposed covalent mechanism in which a glutamate residue is
-phosphorylated; however, further experimentation is necessary to
prove that E384 is phosphorylated and that the phosphorylated enzyme is
kinetically competent to phosphorylate acetate. The complete loss of
activity for the E384D enzyme indicates that displacement of the
active-site nucleophile by a single methylene carbon is sufficient to
completely disrupt catalysis.
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FIG. 2.
CD spectra of unaltered (dotted line) and E384Q (solid
line) acetate kinase from M. thermophila.
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Although the features of the CD spectra were identical, the difference
at 210 nm between the unaltered acetate kinase and
the E384A enzyme was
14% less negative (not shown), suggesting
the possibility that this
replacement may have produced secondary
structural changes. The
thermostability of the E384A kinase was
greater than that of the
unaltered acetate kinase when the enzymes
were preincubated either with
or without ATP (Fig.
3A), a result
which
is consistent with changes in secondary structure. The temperature
stability profile for the unaltered acetate kinase is shifted
approximately 27°C higher in the presence of ATP (Fig.
3A). A
similar
shift was observed on preincubation of the E384A enzyme
with ATP (Fig.
3B), suggesting that ATP interacts with the altered
enzyme in the same
way as it does with the unaltered acetate kinase.
This result is
unlikely if substitution of E384 with alanine produced
conformational
changes in the active site which abolished activity;
however, this
possibility cannot be ruled out. Nonetheless, these
results do not
detract from the results obtained for the E384Q
and E384D enzymes which
indicate that E384 is essential for catalysis.
The small amount of
activity recorded for the E384A enzyme is
unexplained; however, it is
theoretically possible that replacement
with alanine may have increased
the accessibility of substrate
to E385, which functioned as an
active-site nucleophile in place
of E384.
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FIG. 3.
Thermostability profiles of unaltered and E384A acetate
kinase from M. thermophila. The enzyme preparations were
preincubated without ( ) and with ( ) 10 mM ATP (final
concentration) after which samples were incubated for 15 min at the
indicated temperatures, cooled to 4°C, and immediately assayed for
kinase activity. (A) Unaltered acetate kinase. One hundred percent
activity was 470 µmol/min/mg of protein. (B) E384A acetate kinase.
One hundred percent activity was 4.0 µmol/min/mg of protein.
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Mechanistically, an exchange of phosphate between ATP and ADP predicts
a phosphoryl-enzyme intermediate; thus, linking exchange
activity with
kinase activity would provide evidence that both
activities involve the
same intermediate. The authentic and heterologously
produced acetate
kinase from
M. thermophila catalyzed an exchange
of
phosphate between ATP and ADP (Fig.
4),
as reported for other
acetate kinases (
10,
17). Although the
initial rate of exchange
(250 ± 40 nmol/min/mg of protein) was
nearly 3,000-fold lower
than kinase activity (705 ± 70 µmol/min/mg of protein) assayed
under the same conditions, the result
does not rule out the possibility
that a phosphoryl-enzyme intermediate
is required for kinase activity.
Unknown determinants such as substrate
synergism may contribute
to relatively low activity of the partial
reaction. The previously
reported ATP-ADP exchange activity of the
E. coli enzyme is consistent
with a phosphoryl-enzyme
intermediate; however, it was not possible
to exclude the possibility
that kinase activity occurred by a
different mechanism (
14).
Likewise, it cannot be concluded from
the exchange activity alone that
kinase activity for the enzyme
from
M. thermophila involves
a phosphoryl-enzyme intermediate;
however, the loss of kinase activity
on replacement of E384 was
paralleled by the inability to detect
significant (limit of detection,
10 nmol/min/mg of protein) ATP-ADP
exchange activity for any of
the enzymes altered at E384 (data not
shown), which is consistent
with a linkage between the two activities
centering on E384.
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FIG. 4.
ATP-ADP exchange activity of acetate kinase from
M. thermophila. Activity was determined for purified
histidine-tagged () and untagged () M. thermophila
acetate kinase produced in E. coli. The ADP fractions
contained 35,700 () and 92,000 () cpm at equilibrium.
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Further support for involvement of E384 in the active site of the
acetate kinase from
M. thermophila was obtained by replacing
the neighboring E385 residue. E385 is 100% conserved with the
acetate
kinase sequences from diverse microbes (Fig.
1); however,
other
residues with different functionality replaced E385 (Table
2) without
the reduction in activity observed for the enzymes
altered at E384,
suggesting that E385 is not essential for catalysis.
Nonetheless,
replacement of E385 with alanine or aspartate significantly
increased
the
Km values for acetate and ATP, suggesting
that this
residue is important for binding these substrates. However,
the
kinetic constants for the E385Q enzyme were not significantly
different from those for the unaltered acetate kinase, suggesting
that
the negative charge of E385 is inconsequential for substrate
binding.
The unaltered acetate kinase from
M. thermophila was
not
inactivated by NEM (Fig.
5); however, the
E385C enzyme was
inactivated in a time-dependent manner, and
preincubation with
ATP, ADP, acetate, or acetyl phosphate protected
against the inactivation
(Fig.
6), which
suggests that E385 is near the active site.
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FIG. 5.
Time course for NEM inactivation of unaltered and E385C
acetate kinase from M. thermophila. Kinase activity was
determined at the indicated times after addition of NEM to unaltered
( ) or E385C () acetate kinase and compared to a control where no
inhibitor was added to the E385C enzyme ().
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FIG. 6.
Substrate protection from NEM inactivation of the
E385C-altered acetate kinase from M. thermophila. Kinase
activity was determined at the indicated times after addition of NEM to
the E385C acetate kinase preincubated with ADP ( ), ATP ( ),
acetate (), acetyl phosphate (), or no substrate ().
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The acetate kinase isolated from
E. coli is inactivated by
NEM, and the inactivation is protected by preincubation with either
ATP
or ADP, suggesting that a cysteine residue is located near
the active
site (
21). The inhibition is incomplete, and, therefore,
it
was concluded that the cysteine is not involved in catalysis.
Only one
cysteine residue is conserved between the
M. thermophila and
E. coli acetate kinases (Fig.
1); thus, either the
M. thermophila enzyme has no cysteine residue corresponding in
location to the
modified residue in the
E. coli enzyme or
the only conserved cysteine
residue in the
M. thermophila
enzyme (C207) is shielded from attack
by NEM. Preincubation with
acetate or acetyl phosphate does not
protect the
E. coli
acetate kinase from NEM inactivation; however,
the pattern by which
these substrates prevent protection by AMP,
ADP, or ATP suggests that
the binding sites for acetate and acetyl
phosphate are in close
proximity to the phosphoryl binding regions
of ADP and ATP
(
21). The ability of all four substrates to prevent
NEM
inactivation of the
M. thermophila E385C acetate kinase
(Fig.
6) is consistent with the results obtained for the
E. coli enzyme.
Replacement of E97.
Compared with values obtained with the
unaltered acetate kinase, the Km values for
acetate of all the E97-altered enzymes decreased with corresponding
decreases in kcat, which resulted in relatively
smaller differences in the catalytic efficiencies (kcat/Km) (Table 2).
These results suggest that E97 is not essential for catalysis but
influences the binding of acetate. Replacement of E97 with aspartate
was the least effective in lowering the Km for
acetate, suggesting that the negative charge of E97 influences binding
of the acetate anion by charge repulsion. The results also suggest that
displacement of the negative charge by only one methylene carbon
significantly influences the binding of acetate. None of the E97
replacements influenced the Km values for ATP, suggesting no involvement of E97 at the ATP binding site.
The
Km for acetate of the unaltered acetate
kinase is relatively high, which appears to place
M. thermophila at a disadvantage
when competing for substrate; thus,
the ability to decrease the
Km nearly 10-fold by
simple replacement of a single residue suggests
that the enzyme evolved
to prefer a high
Km, which may be
physiologically
significant. Indeed,
M. thermophila and
other
Methanosarcina species
predominate in environments
where the acetate concentrations are
in the millimolar range compared
with species from the acetotrophic
genus
Methanothrix, which
thrive in habitats with acetate concentrations
well below 1 mM
(
22). The acetotrophic
Methanosarcina species
outcompete
Methanothrix species with higher growth rates and
greater
conservation of energy. The acetate-activating enzyme acetate
thiokinase from
Methanothrix soehngenii has a
Km for acetate below
1 mM, which allows growth
of this species in habitats with low
concentrations of acetate where
methanosarcinas are unable to
compete (
9). Apparently, the
M. thermophila acetate kinase
evolved to favor a high
turnover at the expense of a low
Km for
acetate
which supports a faster growth rate in environments with
high
concentrations of acetate.
Replacement of E32 and E334.
The kinetic constants determined
for the E32A and E334A enzymes (Table 2) indicated that neither residue
is essential for catalysis or significantly influences the binding of
acetate or ATP.
Conclusions.
The first site-specific replacement of amino
acids for any acetate kinase has identified a glutamate residue (E384)
in the enzyme from M. thermophila which is essential for
catalysis and is consistent with the previously proposed covalent
mechanism for catalysis of kinase activity. Replacement of two other
glutamates (E385 and E97) suggests that they are involved in substrate
binding.
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ACKNOWLEDGMENTS |
This work was supported by grant DE-FG02-95ER20198 to J.G.F. from
the Department of Energy-Basic Energy Sciences and by National Institutes of Health Individual National Research Service Award 5F32GM16107 to K.S.-W.
We thank Douglas Berg of Washington University, who graciously
accommodated K.S.-W. as a guest in his laboratory, without which this
work could not have been completed. We also thank Madeline Rasche for
valuable discussions and support.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, Pennsylvania State University,
University Park, PA 16802-4500. Phone: (814) 863-5721. Fax: (814)
863-6217. E-mail: jgf3{at}psu.edu.
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Abstract
Introduction
