©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of Intact and Truncated Forms of the Escherichia coli Biotin Carboxyl Carrier Subunit of Acetyl-CoA Carboxylase (*)

(Received for publication, July 18, 1995; and in revised form, December 20, 1995)

Elizabeth Nenortas Dorothy Beckett (§)

From the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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 kK values of 10 times 10^3 and 15 times 10^3M 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.


EXPERIMENTAL PROCEDURES

Chemicals and Biochemicals

All chemicals used in preparation of buffers were at least reagent-grade. Reagent-grade urea from Sigma was recrystallized from 95% ethanol and dried before use in buffer preparation for BCCP purification. Biotinyl-5`-AMP (bio-5`-AMP) was synthesized using the procedure described in Abbott and Beckett (6) . BirA was purified using a previously reported method(6) .

Purification of the Intact BCCP Subunit of the Acetyl-CoA Carboxylase

BCCP was isolated from E. coli strain BMH 71-18 (F`proAB lacI^q lacZDeltaM15 Delta(lac-pro) thi supE) in which BCCP was coexpressed with the biotin carboxylase subunit of E. coli acetyl-CoA carboxylase from plasmid pLS-182(8) . Cells were grown in Luria Bertani (LB) media (9) containing 100 µg/ml ampicillin to an optical density at 600 nm of 1.0. Expression was induced by addition of isopropyl beta-D-thiogalactoside to 0.5 mM and allowed to proceed for 4 h. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5 at 4 °C, 200 mM KCl, 5% glycerol, 0.1 mM dithiothreitol (DTT)), and lysed by sonication. Polyethyleneimine was added to the soluble fraction from lysis to a final concentration of 0.1% (v/v) and the resulting precipitate was washed with buffer containing 600 mM KCl to extract precipitated BCCP. Ammonium sulfate was added to 50% saturation to precipitate the proteins and the recovered precipitate was resuspended in 10 mM potassium phosphate, pH 7.0, 2 M urea, 0.1 mM DTT. This sample was loaded onto a 40-ml HA-Ultrogel (Sepracor) hydroxyapatite column equilibrated in the same buffer. The BCCP eluted in the breakthrough peak in this step and was exchanged into buffer containing no urea by stepwise dialysis against buffers containing successively lower concentrations of the denaturant. The sample was finally exchanged into buffer containing 50 mM potassium phosphate, pH 7.0, 5% glycerol, 0.1 mM DTT and was loaded onto a DEAE-Sephacel (Pharmacia) column equilibrated in the same buffer and the protein was eluted in a linear (0.05-1.0 M) phosphate gradient. The purified protein was exchanged into buffer containing 50 mM Tris-HCl, pH 7.5 at 4 °C, 200 mM KCl, 5% glycerol, 0.1 mM DTT and stored at -80 °C.

Purification of the 87-Residue C-terminal Fragment of the Biotin Carboxyl Carrier Protein Subunit (BCCP87)

Purification of BCCP87 was carried out using a modification of the method described in Chapman-Smith et al.(10) . The protein was overexpressed from plasmid pTM53 in E. coli strain BL21DE3 (Novagen). Modifications of the previously published purification method include resuspension of induced cells in lysis buffer containing 50 mM Tris-HCl, pH 7.5, at 4 °C, 200 mM KCl, 2.5 mM MgCl(2), 1 mM CaCl(2), 5% glycerol. The cleared lysate was brought to a final concentration of 0.2% (v/v) polyethyleneimine. After pelleting the precipitate, DNase I and RNase were added to the supernatant to a final concentration of 40 µg/ml. Digestion proceeded for 2 h at room temperature. Following a 50% (v/v) isopropyl alcohol precipitation, the supernatant was dialyzed against dialysis buffer (50 mM Tris-HCl, pH 7.5, at 4 °C, 0.1 mM DTT) and loaded onto a Q-Sepharose (Pharmacia) column. BCCP87 was eluted using a linear 2-40 mM potassium phosphate gradient, pH 7.0, in 5% glycerol, 0.1 mM DTT. Pooled phosphate gradient fractions were dialyzed against dialysis buffer containing 5% glycerol and loaded onto a second Q-Sepharose column equilibrated in the same buffer. BCCP87 was eluted using a linear 0-0.5 M NaCl gradient in the same buffer. The pure protein was exchanged into buffer containing 50 mM Tris-HCl, pH 7.5, at 4 °C, 200 mM KCl, 5% glycerol, 0.1 mM DTT and stored at -80 °C.

Mass Spectrometry

The molecular weights of BCCP and BCCP87 were determined by MALDI-TOFMS using a Kratos Kompact MALDI III (Shimadzu Scientific, Columbia, MD) equipped with a 337-nm nitrogen laser operating in the linear mode. Samples were exchanged into a 5% (v/v) acetic acid in water by dialysis and then lyophilized to dryness. A 0.3-µl aliquot of 10 µM BCCP or BCCP87 in 0.1% trifluoroacetic acid (v/v) in water:acetonitrile (80:20) was pipetted onto a 0.3-µl aliquot of matrix solution on a stainless steel slide and air-dried prior to analysis. The matrix solutions used for BCCP and BCCP87 were 50 mM 3,5-dimethoxy-4-hydroxycinnamic acid in acetonitrile, 0.1% (v/v) trifluoroacetic acid in water (70:30) and 100 mM alpha-cyano-4-hydroxycinnamic acid in acetonitrile, 0.1% (v/v) trifluoroacetic acid in water (70:30), respectively. Bovine trypsinogen (Sigma) and horse heart cytochrome c (Sigma) were used as calibration standards for BCCP and BCCP87, respectively. Standards were prepared for spectroscopy by dialyzing reconstituted protein samples extensively against 5% (v/v) acetic acid in water and lyophilizing to dryness. Mixtures of the appropriate calibration standard and BCCP or BCCP87 analyte were analyzed as described above and calibration was performed using the known protonated molecular ion (MH) and doubly charged protonated molecular ion (M2H) masses of the internal calibrant which bracketed the analyte of interest.

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(2) (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.

Sedimentation Equilibrium

Equilibrium sedimentation experiments were performed with a Beckman XL-A Optima analytical ultracentrifuge equipped with a four-hole, An-55 rotor. Double sector cells with charcoal-filled epon centerpieces and quartz windows were used for determining protein concentration distributions with the absorption optical system of this instrument. All experiments were performed at 20 °C with samples that were dialyzed extensively against buffer A. Solvent densities were determined pychnometrically. Data were acquired either as a single measurement with nominal spacing of 0.001 cm between radial positions.

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(r) is the concentration of the enzyme at a given radial position, c(m) 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(m) 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.

Steady State Fluorescence Measurements

Fluorescence measurements were made using an SLM 48000 spectrofluorimeter. The sample temperature was maintained at 20.0 ± 0.1 °C with a circulating water bath. The excitation wavelength was 295 nm and emission was monitored from 310 to 450 nm. Excitation and emmission slit widths were set at 4 nm. All measurements were made in the ratio mode using rhodamine B as a quantum counter. Stoichiometric titrations to determine the activities of BCCP and BCCP87 in accepting biotin were performed by sequential addition of small volumes of a concentrated BCCP solution to a solution containing a 1:1 complex of BirA and bio-5`-AMP. The mixtures were allowed to equilibrate for at least 2 min prior to measurement of the spectra. Spectra were corrected for the contribution of buffer and volume changes. The fluorescence intensities utilized in analysis of titration data were obtained by integration of the background corrected fluorescence spectra. No correction was made for the inner filter effect because the extinction coefficients for BCCP and BCCP87 at 295 nm due to tyrosine residue absorbance are nearly zero.

Stopped-flow Fluorescence Measurements

The apparent BirA-catalyzed rates of biotin transfer from bio-5`-AMP to BCCP and BCCP87 were measured using a KinTek Model 2001 stopped-flow instrument. The time-dependent increase in the intrinsic BirA fluorescence was measured upon rapidly mixing a 1:1 complex of BirA and bio-5`-AMP with either acceptor protein. The final concentration of BirA in the observation cell was 1 µM and the concentration of acceptor protein (either BCCP or BCCP87) was varied. The excitation wavelength was set at 295 nm and fluorescence emission was monitored above 340 nm using a cutoff filter (Corion Corp.). Experiments were performed in buffer A. The temperature was maintained at 20 ± 0.1 °C using a circulating water bath. All buffers and solutions were degassed with a stream of argon and filtered through 0.45-µm Acrodisc PTFE filters (Gelman Sciences) before use.


RESULTS

Purification of BCCP and BCCP87

Overexpressed intact BCCP was purified to at least 98% purity as judged by results of SDS-PAGE. The major difficulty encountered in the purification was separation of the BCCP subunit from a second acetyl-CoA carboxylase subunit, the biotin carboxylase. This difficulty arose because efficient overexpression of BCCP required coexpression of biotin carboxylase(8) . Since the two subunits could not be separated using native conditions, the initial steps in the purification were carried out in the presence of 2 M urea. Fortuitously, much of the biotin carboxylase subunit is selectively precipitated in the dialysis step preceeding the first chromatographic step on HA-Ultrogel resin. Additional purification was achieved in this chromatography step since the biotin carboxylase subunit selectively bound to the hydroxyapatite, while BCCP eluted in the wash fraction. The protein was finally purified to homogeneity in native conditions by chromatography on DEAE-Sephacel.

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.

The Molecular Masses of BCCP and BCCP87

The molecular masses of purified BCCP and BCCP87 were determined by MALDI-TOFMS. A representative mass spectrum of BCCP is shown in Fig. 2. The experimentally determined molecular mass for purified BCCP was in good agreement with the average molecular mass calculated from the amino acid sequence(8) , as shown in Table 1. The mass measured using MALDI-TOF differs significantly from the previously reported SDS-PAGE-derived molecular mass of 22.5 kDa(8, 12) . The average molecular mass obtained for purified BCCP87 was in excellent agreement with the value calculated from the amino acid composition (Table 1). The molecular masses obtained for BCCP and BCCP87 indicate that the purified proteins are predominantly in the apo form.


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.





The Assembly States of BCCP and BCCP87

The assembly states of the two proteins were measured by equilibrium sedimentation. A representative scan of the concentration distribution obtained for BCCP87 is shown in Fig. 3. The average molecular weight of BCCP87 estimated by nonlinear least squares analysis using a single species model is 9100 ± 130, a value in good agreement with the analytical molecular weight. The protein is, therefore, monomeric at the concentrations used in this experiment. The average molecular weight determined for BCCP from sedimentation equilibrium data is dependent on rotor speed. The protein was subjected to sedimentation at a loading concentration of 108 µM. Data were acquired at three rotor speeds; 28,000, 22,000, and 16,000 rpm. Analysis of these data sets using a single species model yielded M(r) of 70,000, 109,000, and 182,000, respectively. The agreement of the data with a single species model was poor in all cases. These results indicate that intact BCCP forms a number of large aggregates. Additional experiments using other techniques are required to better determine the assembly properties of this protein.


Figure 3: A, concentration distribution obtained for BCCP87 in buffer A. The loading concentration was 3 times 10M 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.



The Activities of BCCP and BCCP87 in BirA-catalyzed Transfer of Biotin

Two assays were used to determine the activities of the intact and truncated forms of BCCP as substrates in the BirA-catalyzed transfer of biotin from bio-5`-AMP. In the first assay, the activity of the proteins was determined by analysis of the product of the transfer reaction using MALDI-TOFMS. For control and reaction mixtures, acceptor protein was incubated with a catalytic amount of BirA in the absence and presence, respectively, of a 2-fold excess of chemically synthesized bio-5`-AMP over acceptor protein. The mixtures were exchanged into a volatile solvent, lyophilized, and spectra were obtained. The molecular weight of acceptor protein incubated in the presence of bio-5`-AMP should shift quantitatively to the value expected for the modified form of the protein, if the acceptor protein is 100% active in accepting biotin. The experimentally obtained spectra for control and reaction samples of BCCP and BCCP87 exhibited peak shapes consistent with a single protein species. We observed a quantitative shift in molecular mass for both BCCP and BCCP87 to molecular mass values consistent with the biotinated forms of each protein as listed in Table 1. Fig. 4, a MALDI-TOF mass spectrum of an approximately equimolar mixture of BCCP87 control and reaction samples illustrates the resolution obtained between apo and holo forms of the protein.


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 alpha-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.





Kinetics of Transfer of Biotin to BCCP and BCCP87

The rate of transfer of biotin from the adenylate to the two acceptor proteins was measured by stopped-flow fluorescence. The time dependent increase in intrinsic BirA fluorescence intensity obtained upon rapid mixing of apoBCCP87 with a 1:1 complex of BirA and bio-5`-AMP is shown in Fig. 7. Since the equilibrium dissociation constant for the BirA-bio-5`-AMP interaction is approximately 10M(5) in the buffer conditions employed, we are confident that BirA is saturated with bio-5`-AMP prior to mixing with the acceptor protein. As indicated by the trace, the kinetics of biotin transfer to apoBCCP87 are biphasic and nonlinear least squares analysis of the stopped-flow fluorescence data indicates that the process is well described by a double exponential equation. At all concentrations, the amplitude of the total fluorescence change of the stopped-flow data is consistent with that expected for conversion of BirA-bio-5`-AMP to apoBirA. The time constant for the first process is substrate concentration dependent while that of the second is independent of BCCP87 concentration. This is consistent with the first phase reflecting substrate binding followed by chemical transfer of biotin and the second reflecting release of product(s). The following equation applies to this type of multi-step reaction.


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(s) is the equilibrium dissociation constant for binding of apoBCCP to BirA-bio-5`-AMP, k(2) is the rate constant for chemical transfer of biotin from the adenylate to apoBCCP, and k(3) 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(2)/K(s) 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(s)) (Fig. 8A, Table 3). The parameter k(2)/K(s) is, moreover, equivalent to the specificity constant, k/K(m), for enzymatic reactions that adhere to the kinetic mechanism shown in (13) . Values of the individual parameters k(2) and K(s) 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(3), 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(2)/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(2)/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(2)/K(s)(k/K(m)), k(2), and K(s), for the process are shown in Table 3. The constant k(3) 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(m) as well as the individual values of k(2) and K(s) are similar for the intact and truncated forms of BCCP.


DISCUSSION

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 ApoBCCP Subunit and BCCP87 as Models for Study of the BirA-Acceptor Protein Interaction

There is evidence to suggest that apoBCCP, in the absence of the biotin carboxylase and biotin transcarboxylase subunits, provides an adequate model for use in the study of the acceptor protein interaction with BirA-bio-5`-AMP. Propionibacterium shermanii transcarboxylase is the best characterized carboxylase and, analogous to E. coli acetyl-CoA carboxylase, contains the biotin carboxyl, biotin carboxyl carrier, and transcarboxylase functionalities on discrete subunits (14) . Previously, Wood et al.(15) compared the rates of biotination of the P. shermanii biotin-accepting apo-1.3 S subunit and the complete apotranscarboxylase by biotin transcarboxylase synthetase and found that they were approximately the same. We speculate that BirA may catalyze biotin ligation of the BCCP and intact acetyl-CoA carboxylase at similar rates by inference with the P. shermanii holoenzyme synthetase and transcarboxylase results.

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(2)-terminal domain of beta-galactosidase was fused to varying lengths of the COOH-terminal BCCP wild-type sequence. Incorporation of [^3H]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(2)-terminal domain of the protein.

Characterization of the Purified Proteins

We successfully purified the overexpressed BCCP subunit to homogeneity. MALDI-TOFMS analysis confirmed that the molecular mass of the purified BCCP corresponded to that calculated from the sequence and that the purified protein was in the apo form required for study of its interaction with the BirA-bio-5`-AMP complex. As mentioned above, the mass spectrometrically determined molecular mass is approximately 5.7 kDa less than the previously reported SDS-PAGE-derived molecular mass. Li and Cronan (8) proposed that the difference between the calculated mass and that determined from SDS-PAGE is due to the unusually high proline and alanine content between residues 34 and 101. Several lipoated proteins containing Pro/Ala-rich sequences exhibit an analogous discrepancy between sequence calculated and SDS-PAGE-determined molecular masses(8) . Miles et al.(19) have shown that sequential deletion of Pro/Ala sequences from E. coli dihydrolipoamide acetyltransferase results in convergence of the SDS-PAGE- and sequence-derived molecular masses.

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(2) terminus of intact BCCP is involved in protein-protein interactions. By analogy, NH(2)-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) .

The Activities of BCCP and BCCP87

Purified BCCP and BCCP87 are 100% active in the BirA-catalyzed transfer of biotin as measured using two assays. Incubation of either acceptor protein with BirA-bio-5`-AMP results in stoichiometric transfer of biotin to acceptor protein as assessed by mass spectrometric analysis of incubated samples. The result for BCCP87 agrees with the previously reported quantitative incorporation of biotin into BCCP87 determined using mass spectrometry(10) . Fluorescence titrations revealed a stoichiometry of 1:1 for each acceptor protein with the BirA-bio-5`-AMP complex indicating the BCCP and BCCP87 preparations are 100% active in the biotin transfer reaction. We conclude from this result that the assembly of BCCP into large oligomers does not affect its activity in the BirA-catalyzed biotination reaction. The full activity of the two protein preparations moreover makes them suitable for use in measurements of the kinetics of the biotin transfer reaction.

Interaction of BirA-bio-5`-AMP with BCCP and BCCP87

We have used stopped-flow fluorescence to obtain the rate of BirA-catalyzed biotin transfer from bio-5`-AMP as a function of acceptor protein concentration. A simple kinetic model has been used to analyze the data obtained with both the truncated and full-length acceptor proteins. This simple model is justified by the stopped-flow fluorescence results which indicate that the data obtained with both substrates are well described by two exponential phases. The values of k/K(m), K(s), and k(2) obtained for the two proteins indicate that, despite the aggregation of intact BCCP, the activities of the two proteins in the biotin transfer reaction are remarkably similar. The similarity of the parameters obtained for the two forms of the protein justifies the use of the apoBCCP monomer as the unit of concentration in analysis of the kinetic data and lends additional support to the idea that the NH(2)-terminal domain is independent of the COOH-terminal domain in the intact BCCP.

Control of Functional Switching of BirA between Transciptional Repressor and Biotin Ligase

Results of in vivo measurements indicate that in the presence of apo acceptor protein, BirA, in its complex with bio-5`-AMP, functions primarily as a holoenzyme synthetase, the biotin operator is unoccupied, and synthesis of the biotin biosynthetic enzymes is constitutive(7) . Once the cellular requirement for biotin is fulfilled by conversion of acceptor protein from its apo to holo form, the intracellular concentration of BirA-bio-5`-AMP accumulates to a sufficient level to fully saturate the biotin operator and repress transcription of the biotin biosynthetic genes. The switch in BirA-bio-5`-AMP function from a biotin ligase to a transcriptional repressor is thus a consequence of the modulation in the intracellular concentration of this complex. Two kinetic processes are responsible for interconversion of BirA between the unliganded and adenylate bound forms (Fig. 9). Conversion of apoBirA to BirA-bio-5`-AMP is accomplished via the synthesis of bio-5`-AMP from the two substrates biotin and ATP. While the intracellular biotin concentration is very low(23, 24) , the intracellular ATP concentration is in the millimolar range(25) . Biotin is therefore the limiting substrate in synthesis of bio-5`-AMP and the rate of conversion of apoBirA to BirA-bio-5`-AMP is dependent on biotin and apoBirA concentration and k/K(m) for the reaction at saturating ATP concentration. The BirA-bio-5`-AMP complex is kinetically very stable and has a half-life of approximately 30 min at room temperature(5) . It does not, therefore, readily dissociate. Rather, conversion of BirA-bio-5`-AMP to apoBirA occurs solely via transfer of biotin to apoBCCP. The rate of this process depends on k/K(m) for the reaction and the intracellular apoBCCP and BirA-bio-5`-AMP concentrations. According to this model the intracellular ratio of apoBirA to BirA-bio-5`-AMP concentration is constantly modulated in response to changes in intracellular biotin and apoBCCP concentrations. While high apoBCCP and low biotin concentration favor the unliganded form of BirA, high biotin and low apoBCCP concentrations result in an increased concentration of BirA-bio-5`-AMP. Only the adenylate bound form of BirA binds to bioO and an increased concentration of this complex results in increased occupancy of bioO and concomitant transcriptional repression. Moreover, because of the cooperative nature of the BirA-bio-5`-AMP-bioO binding interaction a change in BirA-bio-5`-AMP concentration over a relatively narrow 10-fold range is sufficient to switch transcription of the biotin operon from fully derepressed to fully repressed(6) .


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(m) 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(m) of 1 times 10^7M 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(m) 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.

Summary

In this work we have presented methods for purification of large quantities of the intact apoBCCP and BCCP87 to homogeneity. Both proteins are 100% active in BirA-catalyzed transfer of biotin from bio-5`-AMP. The nearly identical kinetic parameters measured for the two proteins indicate that they are equivalent in their interaction with BirA-bio-5`-AMP. Despite assembly of intact BCCP into large oligomers, the COOH-terminal biotin-accepting domain of intact BCCP appears to behave independently of the NH(2)-terminal domain with respect to the biotin transfer reaction. Monomeric BCCP87 is, therefore, a good model for intact BCCP. Functional switching of BirA between repressor and ligase roles occurs in response to changing intracellular biotin and apoBCCP concentrations. Knowledge of the kinetic parameters for the biotin transfer reaction, obtained in this work, combined with those previously determined for formation of bio-5`-AMP from substrates biotin and ATP and planned studies of the kinetics of BirA association with biotin biosynthetic operator DNA will provide the tools required to quantitatively model partitioning of BirA between its repressor and ligase roles.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM46511 and a DuPont Young Professorship. Instrumentation used for sedimentation and MALDI-TOFMS measurements was supported by National Institutes of Health Grants RR08937 and RR08310, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 410-455-2512; Fax: 410-455-2608.

(^1)
The abbreviations used are: BCCP, biotin carboxyl carrier protein; bio-5`-AMP, biotinyl-5`-AMP; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MALDI-TOFMS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MH, protonated molecular ion; M2H, doubly charged protonated molecular ion.


ACKNOWLEDGEMENTS

We thank Dr. J. E. Cronan, Jr. for the gifts of plasmids pLS-182 and pTM53. We thank the Structural Biochemistry Center at the University of Maryland Baltimore County for helpful advice concerning MALDI-TOFMS.


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