(Received for publication, July 18, 1995; and in revised form, December 20, 1995)
From the
Biotin biosynthesis and retention in Escherichia coli is regulated by the multifunctional protein, BirA. The protein acts as both the transcriptional repressor of the biotin biosynthetic operon and as a ligase for covalent attachment of biotin to a unique lysine residue of the biotin carboxyl carrier protein (BCCP) subunit of the acetyl-CoA carboxylase. Biotinyl-5`-AMP is the activated intermediate for the ligase reaction and the allosteric effector for DNA binding. We have purified and characterized apoBCCP and a truncated form containing the COOH-terminal 87 residues (apoBCCP87). Molecular masses of the proteins measured using matrix-assisted laser desorption ionization time-of-flight mass spectrometry conformed to the expected values. The assembly states of apoBCCP and apoBCCP87 were determined using sedimentation equilibrium ultracentrifugation. Nearly quantitative enzymatic transfer of biotin from BirA-biotinyl-5`-AMP to the apoBCCP forms was assessed using two methods, mass spectrometric analysis of acceptor proteins after incubation with BirA-bio-5`-AMP and a steady state fluorescence assay. The BirA catalyzed rates of transfer of biotin from bio-5`-AMP to apoBCCP and apoBCCP87 were measured by stopped-flow fluorescence. Kinetic parameters estimated from these measurements indicate that the intact and truncated forms of the acceptor protein are functionally identical.
Biotin biosynthesis and retention in Escherichia coli are regulated via a complex set of macromolecular interactions (Fig. 1)(1) . Retention of biotin is dependent on its
covalent ligation to a unique lysine residue of the biotin carboxyl
carrier protein (BCCP) ()subunit of the acetyl-CoA
carboxylase. The multifunctional protein BirA catalyzes formation of an
amide linkage between biotin and the
-amino group of the lysine
residue of BCCP(2) . BirA is also responsible for regulation of
biotin biosynthesis in E. coli at the level of transcription
of the biotin biosynthetic operon(2) . Sequence-specific DNA
binding of BirA to the 40-base pair biotin operator sequence results in
transcriptional repression(3) . The adenylated form of biotin,
bio-5`-AMP, is the activated intermediate in the enzymatic transfer of
biotin to BCCP as well as the positive allosteric effector for binding
of BirA to the biotin operator sequence(3, 4) . BirA
also catalyzes synthesis of the adenylate from the substrates biotin
and ATP(4) . In order to quantitatively assess the
macromolecular interactions that result in biotin biosynthesis and
retention in vivo, our laboratory is engaged in the in
vitro study of the energetics and stoichiometries of the
individual interactions and their coupling.
Figure 1: Scheme of BirA functions that contribute to regulation of biotin biosynthesis and retention in E. coli. I, catalysis of the synthesis of bio-5`-AMP from biotin and ATP. II, catalysis of the covalent linkage of biotin to lysine residue 122 of BCCP. III, sequence-specific binding to the biotin operator sequence to repress biotin biosynthesis.
BirA switches from the
biotin ligation to repressor function in response to the intracellular
requirement for biotin. The apoBCCP concentration serves as the
indicator of the intracellular biotin requirement. If apoBCCP levels
are high, the protein functions as a biotin ligase(1) . Once
the apoBCCP has been converted to the holo form, BirA-bio-5`-AMP
accumulates in the cell to a level sufficiently high for full occupancy
of the biotin operator and concomitant transcriptional repression of
the biotin biosynthetic genes(1) . The quantitative features of
the control of BirA-bio-5`-AMP function are not known. We have
previously reported an equilibrium dissociation constant and a
half-life for the BirA-bio-5`-AMP complex at 20 °C of approximately
10M and 30 min, respectively (5) . The high thermodynamic and kinetic stability of the
complex suggests that in vivo BirA-bio-5`-AMP does not readily
dissociate(5) . We have also measured binding of holoBirA to
bioO using DNase footprint titrations. Results of these measurements
indicate that the binding occurs in the nanomolar range of protein
concentration. The binding mechanism, however, is not simple and
involves cooperative association of two holorepressor monomers with the
two half-sites of the biotin operator(6) . Estimates of the
Gibbs free energies for binding of a holorepressor monomer to a
half-site and the cooperativity term are -9.0 and -2.0
kcal/mol, respectively(6) . No direct measurement of the
interaction of BirA-bio-5`-AMP with apoBCCP is available particularly
since no method has been developed to purify the protein in sufficient
quantities to perform such measurements. The availability of purified
protein, as well as a quantitative method to measure the heterologous
interaction between BirA-bio-5`-AMP and the acceptor protein, will
further contribute to elucidation of the mechanism of control of BirA
function.
In this work we present methods for large-scale
purification of the intact 156-residue BCCP subunit, as well as, an
87-residue COOH-terminal fragment of the protein. It has been
demonstrated that this fragment encodes sufficient information for
biotination in vivo(7) . We have characterized the
molecular masses of the proteins by matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOFMS). Results of
equilibrium sedimentation of the proteins indicate that while the
intact protein forms large oligomers, the 87-residue fragment is
monomeric. The activities of both proteins in accepting biotin have
been demonstrated to be 100% by two methods. Stopped-flow fluorescence
measurements have been used to measure the BirA-catalyzed rates of
biotin transfer from bio-5`-AMP to each biotin acceptor protein.
Measurement of the dependence of the apparent rate on acceptor protein
concentration provides the second order rate constant, kK
, for
BirA-catalyzed transfer of biotin from BirA-bio-5`-AMP to acceptor
protein. Results of these measurements yield k
K
values of 10
10
and 15
10
M
s
for BCCP and
BCCP87, respectively. These results indicate that the COOH-terminal
87-residue BCCP fragment provides a good model for use in study of the
interaction of BirA-bio-5`-AMP with BCCP. The kinetic parameters
presented in this work, as well as previously determined parameters
governing formation of BirA-bio-5`-AMP, have been incorporated into a
model for control of BirA function.
MALDI-TOFMS was also used to
measure the level of BirA-catalyzed biotination of BCCP and BCCP87.
Reactions containing 25 µM BCCP or BCCP87, 0.5 µM BirA, and 50 µM bio-5`-AMP in 10 mM Tris-HCl, pH 7.50 ± 0.10, at 20.0 ± 0.1 °C, 200
mM KCl and 2.5 mM MgCl (buffer A) were
incubated at 20 °C for 30 min. The mixtures were dialyzed
exhaustively against 5% (v/v) acetic acid in water and lyophilized to
dryness. Spectra were acquired as described previously.
Equilibrium sedimentation data of BCCP obtained either at two different rotor speeds or two initial protein concentrations were analyzed in terms of a single, homogeneous species according to the following equation.
where c is the concentration of the enzyme
at a given radial position, c
is the concentration
of the enzyme at some reference position (e.g. the meniscus), M is the molecular weight, v is the partial specific
volume,
is the solvent density,
is the angular velocity, r is the radial position in centimeters from the center of
rotation, r
is the distance in centimeters from
the center of rotation to the meniscus, R is the gas constant, T is the absolute (Kelvin) temperature, and B is a
correction term for a nonzero baseline.
The UV spectrum of BCCP provides additional
evidence for the purity of the preparation. The BCCP sequence contains
no tryptophan, two tyrosine, and four phenylalanine residues.
Consequently the clearly visible phenylalanine fine structure in the UV
spectrum provided confirmation that purified BCCP was free of
contamination both from nucleic acids and tryptophan-containing
proteins. An extinction coefficient of 2900 liter mol cm
at 276 nm was calculated for BCCP using the
method of Gill and von Hippel (11) . The yield of protein was
1.3 mg/g of wet cell paste.
The procedure used for purification of
BCCP87 overexpressed in E. coli was a modification of a method
developed by Chapman-Smith et al.(10) . This earlier
procedure yielded preparations that contained 2-4% nucleic acid
contamination(10) , a level that precludes accurate
determination of the protein concentration. Therefore, several
additional steps were added to the procedure to ensure protein purity.
BCCP87 was at least 97% pure as judged by Coomassie staining of samples
electrophoresed on 16% SDS-PAGE gels. As with intact BCCP, the BCCP87
UV spectrum was used to assess purity due to its aromatic amino acid
content of one tyrosine and two phenylalanines per chain. The presence
of phenylalanine fine structure indicated the preparation was free of
contaminating nucleic acids. An extinction coefficient of 1450 liter
mol cm
at 276 nm was calculated
for BCCP87 using the method of Gill and von Hippel(11) . The
total yield of the protein per gram of wet cell paste was 2.2 mg.
Figure 2: MALDI-TOF mass spectrum of purified BCCP. The spectrum was obtained using a Kratos Kompact MALDI III mass spectrometer equipped with a 337-nm nitrogen laser. Aliquots of 0.3 µl of 50 µM BCCP and internal calibrant bovine trypsinogen in 0.1% trifluoroacetic acid in water:acetonitrile (80:20), and 0.3 µl of 100 mM 3,5-dimethoxy-4-hydroxycinnamic acid in acetonitrile, 0.1% trifluoroacetic acid in water (70:30), were deposited on a metal slide and air-dried prior to analysis.
Figure 3:
A,
concentration distribution obtained for BCCP87 in buffer A. The loading
concentration was 3 10
M and the
rotor speed was 32,000 rpm. A calculated value of the partial specific
volume used in analyzing the data was 0.73 ml/mg(31) . The solid line represents the best fit of the data obtained by
nonlinear least squares analysis using a single exponential model. B, residuals of the fit.
Figure 4:
MALDI-TOF mass spectrum of an equimolar
mixture of apoBCCP87 and holoBCCP87. Mass spectrometry conditions are
the same as in Fig. 2except 50 mM -cyano-4-hydroxycinnamic acid was used as the matrix and
horse heart cytochrome c was used as the external
calibrant.
The dependence of intrinsic BirA fluorescence intensity on bio-5`-AMP ligation state was also used to assay the activity of the BCCP preparations. As shown in Fig. 5, addition of an equimolar amount of bio-5`-AMP to BirA results in an approximately 40% decrease in the intrinsic BirA fluorescence intensity. Titration of the BirA-bio-5`-AMP complex with excess apoBCCP87 results in the transfer of biotin and concomitant return of the spectrum to that of unliganded BirA (Fig. 5). Measurement of the dependence of the return on BCCP or BCCP87 concentration provides a measure of the activity of either protein in accepting biotin. If the acceptor protein is 100% active in accepting biotin from BirA-bio-5`-AMP, then a breakpoint titration of BirA-bio-5`-AMP with acceptor protein should yield a 1:1 stoichiometry. An excitation wavelength of 295 nm was used so that neither BCCP nor BCCP87, which lack tryptophan residues, contribute to the fluorescence emission spectrum.
Figure 5: Steady state fluorescence titration of 2.0 µM BirA-bio-5`-AMP with excess BCCP87 in buffer A at 20 °C. a, [BirA] = 2.0 µM; b, [BirA-bio-5`-AMP] = 2.0 µM; c, [BirA] = 2 µM and [BCCP87] = 13 µM.
A representative breakpoint titration of BirA-bio-5`-AMP with BCCP is shown in Fig. 6. Analysis of the stoichiometric titration data is tabulated in Table 2for BCCP and BCCP87. These results indicate that the intrinsic fluorescence of the unliganded form of BirA is regained upon addition of 1 mol of BCCP or BCCP87 per mol of BirA-bio-5`-AMP. Based on these results, the activities of both proteins in accepting biotin are 100%.
Figure 6: Titration of 2.0 µM BirA-bio-5`-AMP with BCCP. The solid line represents the best-fit curve obtained from nonlinear least squares analysis of the data using a simple relation for stoichiometric binding of BCCP to BirA-bio-5`-AMP.
Figure 7: Stopped-flow fluorescence measurement of BirA-catalyzed transfer of biotin from bio-5`-AMP to apoBCCP87. The experiment was performed using a KinTek SF-2001 stopped-flow instrument with an excitation wavelength of 295 nm. The emission was monitored above 340 nm using a cutoff filter. The reaction was performed in buffer A at 20 °C with a [BirA-bio-5`-AMP] of 1.0 µM and a [BCCP87] of 175 µM. The solid line is the best fit curve obtained from nonlinear least squares analysis of the data using a double exponential model.
where BCCP refers to either acceptor protein, K is the equilibrium dissociation constant for binding of apoBCCP
to BirA-bio-5`-AMP, k
is the rate constant for
chemical transfer of biotin from the adenylate to apoBCCP, and k
is the rate constant governing release of the
product(s) from the enzyme-product complex. The relationship between
the apparent rate of the first phase in the time course, k
, under pseudo-first order conditions where
[BCCP]
[BirA-bio-5`-AMP], to apoBCCP
concentration (14) is shown below.
Based on this relationship, k/K
can be obtained from the
slope of the dependence of the apparent rate of the first phase in the
reaction on substrate concentration at low substrate concentrations
([BCCP]
K
) (Fig. 8A, Table 3). The parameter k
/K
is, moreover, equivalent to the specificity constant, k
/K
, for enzymatic
reactions that adhere to the kinetic mechanism shown in (13) . Values of the individual parameters k
and K
for biotin transfer
were also estimated from nonlinear least squares analysis of the data
over the entire range of apoBCCP87 concentration (Fig. 8A and Table 3). The rate constant governing product release, k
, was obtained directly from nonlinear least
squares analysis of the second phase in the stopped-flow data (Table 3).
Figure 8:
A,
dependence of k on [BCCP87]. The solid line was simulated using parameters obtained from
nonlinear least squares analysis of the data using . Each
point represents the average of 6-10 independent measurements of
the rate at a given substrate concentration. The dashed line corresponds to the linear portion of the curve where
[BCCP87]
K
. The slope of
this line gives k
/K
,
which is equivalent to the specificity constant k
/K
. B,
dependence of k
on [BCCP]. The solid line was simulated using parameters obtained from
nonlinear least squares analysis of the data using . Each
point represents the average of 6-9 independent measurements of
the rate at a given substrate concentration. The dashed line corresponds to the linear portion of the curve where
[BCCP]
K
. The slope of
this line gives k
/K
,
which is equivalent to the specificity constant k
/K
.
The apparent rate of BirA-catalyzed transfer of
biotin from bio-5`-AMP to apoBCCP was also measured by stopped-flow
fluorescence. The time course of this reaction is also biphasic and is
well described by a double exponential equation. The amplitude and rate
constants for the second phase, however, varied with apoBCCP
concentration. We believe that this is due to the presence of an
artifact in the stopped-flow data at increased substrate concentrations
resulting from the tendency of the large aggregates of apoBCCP to
scatter light. The experimental support for this interpretation is
based on the observation that at low apoBCCP concentrations the
amplitude of the total change in the fluorescence intensity is
consistent with that obtained in transfer of biotin to monomeric
apoBCCP87. At higher apoBCCP concentrations, however, the final
asymptotic limit of the fluorescence intensity increased with
increasing substrate concentration. The values of k for the first kinetic phase were obtained over a range of apoBCCP
concentration (Fig. 8B). The resolved kinetic
parameters, k
/K
(k
/K
), k
, and K
, for the process are
shown in Table 3. The constant k
could not
be obtained for the second phase in transfer of biotin to intact
apoBCCP because of the artifact discussed above. Values of k
/K
as well as the
individual values of k
and K
are similar for the intact and truncated forms of BCCP.
In order to measure the interaction of BirA-bio-5`-AMP with apoBCCP, BirA and apoBCCP must be purified in quantities sufficient to perform biophysical studies. Our laboratory has previously reported a large scale purification of BirA(6) . The BirA preparations have been characterized as 100% active in both binding to bio-5`-AMP using fluorescence titration and binding to bioO using the nitrocellulose filter binding technique(6) . In vivo, BirA catalyzes the covalent attachment of biotin to lysine 122 of the BCCP subunit of acetyl-CoA carboxylase. Both the overall subunit stoichiometry of acetyl-CoA carboxylase in vivo, as well as the assembly state of BCCP, which is relevant for the biotin transfer reaction in vivo, are unknown. Although protocols for purification of holoBCCP are available in the literature, this work contains the first report of a method for obtaining large quantities of homogeneous preparations of apoBCCP. A method for purification of apoBCCP87 has been recently published which yields protein containing significant amounts of nucleic acid contamination(10) . The preparations obtained using the protocol outlined in this work are homogeneous as judged by the quality of their UV spectra. As indicated under ``Results,'' this is a particularly good indicator of the purity of apoBCCP and apoBCCP87 preparations because of the aromatic amino acid compositions of these proteins. The availability of large quantities of homogeneous preparations of apoBCCP and apoBCCP87 allows direct measurement of the interaction of BirA-bio-5`-AMP with both proteins.
The in vivo activities of the biotinated domains from a
number of biotin acceptor subunits provide evidence that the domain
alone is a good model for use in study of the interaction between
BirA-bio-5`-AMP and BCCP. Li and Cronan (8) cloned the E.
coli BCCP subunit and constructed a series of fusion proteins in
which the NH-terminal domain of
-galactosidase was
fused to varying lengths of the COOH-terminal BCCP wild-type sequence.
Incorporation of [
H]biotin into fusion proteins
containing at least the COOH-terminal 84 amino acid residues of BCCP
was catalyzed by BirA in vivo(8) . Therefore, the
COOH-terminal domain of BCCP, in the absence of the other acetyl-CoA
carboxylase subunits, is sufficient for biotination by BirA in
vivo. In like manner, Cronan (7) constructed fusion
proteins using the biotin-accepting 1.3 S subunit of P. shermanii transcarboxylase instead of BCCP. The 1.3 S subunit fusion
proteins were expressed in E. coli and found to be biotinated
by cellular BirA(7) . Therefore, the biotin accepting domain of
the P. shermanii 1.3 S subunit is also sufficient for
biotination by BirA in the absence of the other transcarboxylase
subunits. Accessibility of the biotin acceptor domain of a carboxylase
is required for enzymatic transfer of biotin from the holoenzyme
synthetase and for its function within the intact carboxylase.
Brocklehurst and Perham (16) noted the resemblance between the
Pro-Ala-Pro containing sequences of the carboxylases and the flexible
linkages of lipoyl domains to dehydrogenases. Pro/Ala lipoyl domain
linkages of dehydrogenases have been studied by NMR spectroscopy and
determined to be extremely mobile(17, 18) . Both the
biotinylated domains of carboxylases and the lipoylated domains of
dehydrogenases contain prosthetic groups attached via an amide linkage
to a unique lysine residue of the domain. Both biotin and lipoate
prosthetic groups require sufficient mobility to migrate between
discrete enzymatic domains of the carboxylases and dehydrogenases,
respectively. Due to the inherent flexibility required of a biotin
containing domain within an assembled carboxylase, the COOH-terminal
domain of the BCCP is probably somewhat independent of the
NH
-terminal domain of the protein.
Measurements of the interaction of BCCP with BirA require knowledge of the assembly state of each protein. Sedimentation equilibrium studies of purified apoBCCP revealed that it self-associates to a 4-10-mer in the micromolar range of concentration. These findings agree with the sedimentation equilibrium results of Fall and Vagelos(12) . Fall and Vagelos (12) reported that dilute solutions of BCCP (0.2 to 0.5 mg/ml), obtained from purifying intact E. coli acetyl-CoA carboxylase, are polydisperse systems with molecular masses ranging from 20 to 200 kDa. Interpretation of the results of equilibrium measurements of protein-protein associations, in which one of the proteins is polydisperse, is complex. Use of a BCCP species which has a single assembly state over a broad concentration range is desired. Nervi et al.(20) purified an 82-residue protein containing the biotin domain of BCCP. Sedimentation velocity analysis of this protein yielded a molecular weight of 9100 at concentrations as high as 8 mg/ml, which is consistent with a monomeric assembly state(20) . Subsequent sequencing of the 82-residue protein by Sutton et al.(21) , and comparison to the deduced BCCP amino acid sequence (8) confirms that the characterized BCCP fragment corresponds to the COOH-terminal 82 residues of the intact BCCP subunit. In order to have a monomeric acceptor protein species available, we purified the protein corresponding to the COOH-terminal 87 residues of BCCP, BCCP87, to apparent homogeneity.
The reported
MALDI-TOF molecular mass of purified BCCP87 agrees with the
sequence-calculated molecular mass for the apo form and with the value
reported by Chapman-Smith et al.(10) . Sedimentation
equilibrium results for BCCP87 show that it is monomeric in solution.
Comparison of the assembly state results for BCCP and BCCP87 indicates
that the NH terminus of intact BCCP is involved in
protein-protein interactions. By analogy, NH
-terminal
residues 1-18 of the biotin containing 1.3 S subunit of P.
shermanii transcarboxylase are required for assembly of
catalytically active transcarboxylase(22) .
Figure 9: Interconversion of BirA between unliganded and activated adenylate-bound forms. Synthesis of bio-5`-AMP from substrates biotin and ATP converts apoBirA to the activated BirA-bio-5`-AMP form. When bound to bio-5`-AMP, BirA acts as either a transcriptional repressor or biotin ligase. In the presence of sufficient apoBCCP, BirA-bio-5`-AMP catalyzes transfer of biotin to apoBCCP. Initial activation of BirA depends on the intracellular biotin concentration, while conversion of BirA-bio-5`-AMP to unliganded BirA depends on the apoBCCP intracellular concentration.
Consistent with the
kinetic model discussed above, repression of transcription of the
biotin biosynthetic operon is directly related to the intracellular
half-life of BirA-bio-5`-AMP. The kinetic parameters determined in this
work as well as those previously determined for synthesis of bio-5`-AMP
from ATP and biotin (5, 26) can be used to predict the
half-lives of both forms of BirA. The rate of conversion of apoBirA to
BirA-bio-5`-AMP is equal to the product of k/K
for the synthesis of
bio-5`-AMP at saturating [ATP] and the apoBirA and biotin
concentrations. A pseudo-first order rate constant for the process can
be calculated from the estimated value of k
/K
of 1
10
M
s
and an
estimated intracellular biotin concentration of 10 nM. This
rate constant is 0.1 s
and the resulting estimated
half-life of apoBirA is approximately 6 s. Based on these numbers and
the reasonable assumption that biotin concentration is in the nanomolar
range(23, 24) , we conclude that conversion of apoBirA
to holoBirA is rapid. The rate of depletion of the holoBirA pool to
apoBirA via the biotin transfer reaction is equal to the product of k
/K
for the transfer
reaction and the concentrations of holoBirA and apoBCCP. The
bimolecular rate constant for the reaction determined in this work is
approximately 15,000 M
s
. Again, a pseudo-first order rate constant
for the process can be calculated from this bimolecular rate constant
and [apoBCCP]. The calculated rate constant and half-life for
the conversion of holoBirA to apoBirA at 1 micromolar apoBCCP are 0.015
s
and 46 s, respectively. The magnitudes of these
values are, of course, directly dependent on apoBCCP concentration and
lower acceptor protein concentrations result in longer half-lives for
holoBirA. The in vivo apoBCCP concentration has, in fact, been
estimated to be considerably lower than 1 micromolar(27) . The
half-life of holoBirA is, in turn, directly related to the level of
repression of the biotin biosynthetic operon since the probability of
formation of the holoBirA-bio complex increases as the BirA-bio-5`-AMP
half-life increases. Our ability to quantitatively predict the
relationship between this half-life and the formation of the
protein-DNA complex, however, is hindered by the absence of
measurements of the kinetics of association of BirA-bio-5`-AMP with
bioO. This association process could, in principle, occur by a complex
mechanism involving initial association of the repressor with
nonspecific DNA followed by sliding or hopping to the specific sequence (28, 29, 30) and concomitant cooperative
dimerization(6) . The short half-life for conversion of apoBirA
to holoBirA is significant since it favors formation of the
``active'' form of BirA for both site-specific DNA binding
and biotin transfer. The relatively long half-life of the
BirA-bio-5`-AMP complex may be critical for the ability of bioO to
effectively compete with apoBCCP at levels of the acceptor protein that
are consistent with a low metabolic biotin requirement.