tiie journal of biological chemistry 269. no. 43, 28 ... · water, 5 ml of binding buffer, and 3 ml...

TiiE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 269. No. 43, Issue of October 28, pp. 27051-27056,1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Kinetics and Regulation of Pantothenate Kinase from Escherichia coli*

(Received for publication, February 28, 1994)

Woo-Joo Son& and Suzanne JackowskiO From the Department of Biochemistry, St. Jude Children$ Research Hospital, Memphis, lknnessee 38101 and the Depar~ment of B i ~ h e m i s ~ ~ , ~ ~ i v e r s i ~ y of Iknnessee, ~ e m p h ~ s , Tennessee 38163

Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis and is regulated by feedback inhibition by CoA. Pantothenate kinase was purified to homogeneity from Escherichia coli and was shown to exist as a homodimer. Kinetic analysis indicated the presence of two ATP binding sites that exhibited positive cooperativity with a Hill coefficient of 1.46. Site-directed mutagenesis of lysine 101 to methionine (KlOlM) resulted in the inactivation of the enzyme, although dimer formation was not altered The KlOlM mutant was unable to bind either adenosine 5’-0-(3-thiotri- phosphate) or CoA, supporting the conclusion from kinetic analysis that both the substrate and inhibitor bind to the same site on the enzyme. CoA binding was not cooperative. Coexpression of the KlOlM mutant gene on a high copy number plasmid in the presence of a chromosomal copy of the wild-type gene resulted in the production of heterodimers between active and inactive subunits. Kinetic analysis of the chimeric heterodimers showed the absence of cooperative ATP interactions and indicated a sequential kinetic mechanism for pantothenate kinase with ATP binding first and pantothenate second. Thus, pantothenate kinase regulation involves the competitive binding of CoA to the ATP site, which blocks ATP binding at one site and prevents positive cooperative ATP binding to the second site on the dimer.

Pantothenate kinase (ATPo-pantothenate 4’-phosphotrans- ferase, EC 2.7.1.33) is an essential protein that catalyzes the phosphorylation of the vitamin pantothenate at the 4‘ position to form phosphopantothenate. The formation of phosphopantothenate is the first committed step in the universal biosynthetic pathway leading to CoA. Phosphopantothenate is rapidly me- tabolized to CoA (for review, see Magnuson et al. (199311, which participates as an acyl group carrier in the tricarboxylic acid cycle, fatty acid metabolism, and numerous other reactions of intermediary metabolism (for reviews, see Abiko (1975) and Dawes and Large (1982)). Escherichia coli is capable of synthe- sizing pantothenate de novo, and a sodium-dependent per-

GM 34496, Cancer Center Support (CORE) Grant CA 21765, and the * This work was supported by National Institutes of Health Grant

American Lebanese Syrian Associated Chanties. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTMIEMBL Data Bank with accession number(s) M90071. The nuc2eotide sequenceis) reported in this paper has been submitted

Genetics, University of Michigan Medical School, Ann Arbor, Michigan $ Present address: Howard Hughes Medical Inst., Dept. of Human

48109. 5 To whom correspondence should be addressed: Dept. of Biochemis-

Memphis, TN 38101. Tel.: 901-522-0494; Fax: 901-525-8025. try, St. Jude Children’s Research Hospital, 332 North Lauderdale,

mease exists in both bacteria (Vallari and Rock, 1985a, 1985b; Jackowski and Mix, 1990) and mammals (Barbarat and Po- devin, 1986; Lopaschuk et d., 1987) that is responsible for active transport of pantothenate into the cell. However, metabolic labeling experiments in E. coli (Jackowski and Rock, 1981) and in rat heart (Robishaw et ai., 1982; Robishaw and Neely, 1984) show that the utilization rather than the supply of pantothenate controls the rate of CoA biosynthesis. In fact, wild-type E. coli produces 15-fold more pantothenate than is required for maintaining the intracellular CoA level, and the excess pantothenate i s excreted from the cells (Jackowski and Rock, 1981).

Pantothenate kinase activity in E. coli is regulated by feedback inhibition by CoA and its thioesters (Vallari et al., 19871, which also have negative regulatory effects on pantothenate kinases from mammals (Fisher et al., 1985; Robishaw and Neely, 1985) and plants (Falk and Guerra, 1993). Nonesterified CoA is the most potent inhibitor in vitro of the enzyme from E. coli, although acetyl-coA, which constitutes the major propor- tion of the CoA pool under most growth conditions, is 20% as effective (Vallari et al., 1987). A carbon source shift experiment confirmed that n o n e s ~ r i ~ e d CoA is also a more potent inhibitor of pantothenate kinase activity in vivo (Vallari et al., 1987). CoA inhibition is competitive with ATP, suggesting that both ligands occupy the same site on the protein and providing a mechanism for the c ~ r ~ n a t i o n of pantothena~ kinase activity with the energy state of the cell (Vallari et ai., 1987).

E. coli mutants with a temperature-sensitive pantothenate kinase activity are also temperature sensitive for CoA synthe- sis and growth (Vallari and Rock, 1987). The E. coli gene en- coding pantothenate kinase was recently cloned using func- tional complementation of a temperature-sensitive allele, coaAlti(Ts) (Song and Jackowski, 1992). DNA sequence analysis of the c o d gene corresponding to pantothena~ kinase re- veals a single open reading frame that encodes two pantothenate kinase proteins from independent translational start sites. Amino-terminal sequence determinations of gel-purified protein confirmed that the smaller protein (35.4 kDa) lacked 8 amino acids that are present at the amino terminus of the larger pantothenate kinase isoform (36.4 kDa). Strains with multiple copies of the c o d gene produced as much as 76-fold- higher specific activities of pantothenate kinase in uitro; however, there was only a 2.7-fold increase in the steady-state level of CoA in vivo. Thus, large alterations in the expression of the c o d gene are not reflected by a similar change in the intracellular CoA content. This is attributed to stringent feedback regulation of pantothenate kinase by CoA, but inhibition of the 4’-phosphopantetheine adenylyltransferase step also contrib- utes to limiting the expansion of the CoA pool when pantothenate kinase is overexpressed (Vallari and Jackowski, 1988; Song and Jackowski, 1992). These data point to allosteric regulation of pantothenate kinase activity, as opposed to gene expression, as the critical factor in regulating the cellular CoA

27051

27052 Pantothenate Kinase from E. coli

content. Virtually nothing is known about the biochemistry or kinetic mechanism of this ubiquitous key regulatory enzyme; therefore, we have purified pantothenate kinase to homogeneity to define its subunit structure and kinetic mechanism.

EXPERIMENTAL PROCEDURES Materials-Sources of supplies were as follows: Amicon, Centricon-30

concentrator; Bio-Rad, Mi-Gel 10, Mx-Gel Hz hydrazide, and Bradford dye-binding protein assay reagents; DuPont NEN, P-[3-3H]alanine (specific activity 91.5 Ci/mmol), [35S]ATPyS1 (specific activity 1457 Ci/ mmol), and ~-[l-'~Clpantothenate (specific activity 57.4 Ci/mol); Phar- macia Biotech Inc., Blue Sepharose CL-GB resin and Superose-12 column; Promega, altered sites in vitro mutagenesis kit; Sigma, antibi- otics, ATP, CoA, and protein standards; and Whatman, DE81 filter discs. C3H1CoA was prepared by metabolically labeling strain SJ16 (panD2) with P-[3-3Hlalanine and isolation of the c3HlCoA by ion-ex- change chromatography (Jackowski and Rock, 1981). All other materials were reagent grade or better.

Bacterial Strains and Plasmids-The E. coli K-12 strains used in this work were strain UB1005 (meti31 relAl spoTl gyrA216 A- hR F-) (Booth, 1980) and strain DV73 (codlS(Ts) metBl relAl spoTl gyrA216 relAl sr1::TnlO A hR F-). Media were LB and LB solidified with 1.5% agar (Miller, 1972) containing, when necessary, ampicillin (100 pg/ml), ka- namycin (50 pg/ml), or tetracycline (15 pg/ml). Plasmid pWS7-13-2 contained the c o d gene as described previously (Song and Jackowski, 1992), and plasmid pGP1-2 encoded T7 RNA polymerase (Tabor, 1990).

Construction of the KlOlM Mutant-Mutagenesis was performed by following the procedures recommended by the manufacturer using the Altered Sites in vitro mutagenesis system (Promega). Briefly, the EcoRI-PstI fragment of pWS7-13-2 was transferred into EcoRI-PstI- digested pAlter-1, resulting in a Amps TetR plasmid. The rescued single- stranded pWS9 was annealed with the ampicillin repair oligonucleotide (Promega) and the 5'-end phosphorylated mutagenic oligonucleotide (5'-CGCGGTGGGGATAAGTACAACCGCC-3'; the two mutated nucleotides are underlined), resulting in plasmid pK101M. Plasmid pBS- KlOlM was constructed by transferring the 1.57-kilobase EcoRI-SphI fragment of pKlOlM into the EcoRI-SphI-digested pBS(+) (Stratagene). The presence of only this specific mutation in the pantothenate kinase sequence was confirmed by DNA sequencing. The GenBank accession number for the nucleotide sequence of coaA is M90071.

Construction of pETKlO1M and Isolation of HisTag-KlOlM Protein-The 1.24-kilobase AcyI-PstI fragment from pBS-KlO1M was blunt ended and ligated with the blunt ended BamHI-digested PET-15b (Novagen) resulting in PET-KlOlM. HisTag-KlO1M protein was overproduced from strain BL21(DE3)/pET-KlOlM by the addition of 0.4 mM isopropyl-1-thio-P-D-galactopyranoside at a cell density of 2 x lo8 cells/ ml. Following 4 h of incubation at 37 "C, cells (20 ml) were harvested and resuspended in 700 p1 of binding buffer (1 mM imidazole, 0.5 M

NaCl, 20 mM Tris-HC1, pH 7.9). The cell extract was prepared by 1 min of sonication and 5 min of centrifugation in a microcentrifuge. The protein extract (750 mg in 400 pl of binding buffer) was mixed with 100 pl of Ni2+-charged HisTag resin (Novagen) by rotating at 10 rpm for 20 min at 4 "C. The mixture was centrifuged at 1,500 x g for 1 min, and the supernatant was saved. The resin was successively washed with 1 ml of binding buffer, followed by 1 ml of 60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HC1, pH 7.9. The bound protein was eluted with 600 pl of elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HC1, pH 7.9). The column was stripped with 300 pl of 100 mM EDTA, 0.5 M NaCl, 20 mM Tris-HC1, pH 7.9. The supernatants from each step were saved to measure pantothenate kinase activity and protein level by immunoblotting. Ni2+- charged resin was regenerated by treating 1 ml of the resin with 3 ml water, 5 ml of binding buffer, and 3 ml of 50 mM NiSO,.

Pantothenate Kinase Purification-Protein preparation steps were performed at 4 "C. Pantothenate kinase protein was overexpressed in strain UBlOO5/pWS7-13-2/pGP1-2 by heat induction of T7 RNA polymerase as described by Tabor (1990). Cells from a 200-ml culture were harvested, and the cell pellet was resuspended in lysis buffer (0.2 g celVml, 50 mM Tris-HC1, pH 7.4, 1 mM MgCl,). The cells were broken in a French press at 18,000 pounddsquare inch, and the protein precipi- tated between 35 and 60% ammonium sulfate was dialyzed against 0.1 M Tris-HC1, pH 7.4.

Two methods were used to prepare pure pantothenate kinase. The dialyzed ammonium sulfate preparation (-21 mg of protein in 3.1 ml of 50 mM Tris-HC1, pH 6.5) was loaded onto a Blue Sepharose CL-GB

The abbreviation used is: ATPyS, adenosine 5'-043-thiotriphos- phate).

column (11.5 ml, 1.5 x 6.5 cm) that was equilibrated with the starting buffer (50 m Tris-HC1, pH 6.5). The column was washed with 20 ml of starting buffer followed by 30 ml of 50 m Tris-HCI, pH 7.4. Pantothe- nate kinase was eluted with 30 ml of 0.25 mM CoA in 50 m~ Tris-HC1, pH 7.4. One-pl aliquots from the 3.7-ml fractions were assayed, and those fractions with high levels of pantothenate kinase activity were pooled and concentrated using a Centricon-30 concentrator (molecular weight cut-off 30 kDa) at 5,000 x g.

Anti-pantothenate kinase antibodies were raised from rabbits with the protein purified by Blue Sepharose chromatography. The IgG serum fraction was isolated, and monospecific pantothenate kinase antibodies were purified by affinity chromatography on a column prepared by coupling purified pantothenate kinase protein to Mi-Gel 10 gel (Harlow and Lane, 1988). The purified antibody was in turn coupled to Mi-Gel Hz hydrazide gel as recommended by the manufacturer to prepare the immunoaffinity column used to purify pantothenate kinase.

Pantothenate kinase protein was purified in one step by immunoaffinity chromatography. The total cell extract (1.6 mg) was dialyzed against 10 mM Tris-HC1, pH 7.5, and loaded onto the immunoafinity column (2 ml, 1 x 2.5 cm), and 2-ml fractions were collected. The column was washed with 40 ml of 10 mM Tris-HC1, pH 7.5, and then with 40 ml of 0.5 M NaCl in 10 mM Tris-HC1, pH 7.5. Pantothenate kinase was eluted with 20 ml of 0.1 M triethylamine, pH 11.5, into tubes containing 0.5 ml of 1 M Tris-HC1, pH 8.0, to immediately neutralize the triethylamine. Rapid neutralization was necessary because prolonged exposure to pH 11.5 inactivated the enzyme. The fractions with high enzyme activity as determined by an assay of 10-pl aliquots were pooled, concentrated with a Centricon-30, and dialyzed against 100 mM Tris-HCl, pH 7.4. The protein concentration was determined by the Bio-Rad ver- sion of Bradford's dye-binding assay (Bradford, 1980) with bovine serum albumin as a standard. However, for measuring the moles of purified protein in the determination of the number of binding sites and other kinetic properties, an absorbance measurement at A,,, was used. The dimer molar extinction coefficient ( E = 87,660) was calculated as described by Perkins (1986).

Gel Filtration Chromatography-Pantothenate kinase (1 pg) purified by Blue Sepharose chromatography was loaded onto a Superose-12 column (Pharmacia) equilibrated with running buffer (0.2 M KC1 in 50 m Tris, pH 7.5) and developed at the rate of 0.3 mumin at 4 "C. The 30-8 fractions were collected, and the pantothenate kinase activity of 10-pl aliquots from each fraction was measured to determine the elution volume of the protein. The elution volume of inactive pantothenate kinase was determined by SDS-polyacrylamide gel electrophoresis and immunoblotting of the fractions. The elution volumes of protein standards were detected by optical absorbance at 280 nm. Protein standards (Sigma) were as follows: aldolase (161 kDa); alcohol dehydrogenase (150 kDa); bovine serum albumin (66 kDa); ovalbumin (43 kDa); carbonic anhydrase (29 kDa); myoglobin (17.5 m a ) ; cytochrome c (12.4 m a ) . Blue dextran (2,000 kDa) and tyrosine (0.18 kDa) were used to determine the void volume (V,, 7.7 ml) and the exclusion volume (V,, 21.7 ml), respectively.

Sedimentation Velocity-Protein standards (1 mg) and purified pantothenate kinase (5 pg) were layered on the top of 5-20% sucrose gradients (total 11 ml) in 50 IMI Tris-HC1, pH 7.5, and 0.2 M KCl. After 23 h of centrifugation at 200,000 x g at 4 "C in a SW41 Ti rotor in a Beckman L8-70 ultracentrifuge, the gradients were fractionated (total of 65, 170-p1 fractions) using a peristaltic pump. The absorbance at 280 nm for protein standards and enzymatic activity for pantothenate kinase were measured to localize the proteins in the fractions. The protein standards used were yeast alcohol dehydrogenase (150 kDa), bovine serum albumin (66 m a ) , and ovalbumin (43 kDa).

Equilibrium Dialysis-Equilibrium dialysis was performed using an equilibrium microvolume dialyzer (Hoefer Scientific) with a diaylsis membrane (molecular weight cut-off 12,000-14,000) rotating at 20 rpm at 25 "C. Fifty pmol (final concentration, 0.5 w) of pantothenate kinase protein in 100 pl of dialysis buffer (100 nm Tris-HC1, pH 7.4, 2.5 mM MgCl,) was loaded on one side of a well, and various concentrations of [35S]ATPyS (0.8-2.6 F, specific activity 14 CUmmol) or t3HlCoA(2.5-30 m, specific activity 265 mCi/mmol) in the same buffer were loaded on the other side of individual wells. Bovine serum albumin was added to both wells to a final concentration of 1.8 w. The module was rotated for 12-15 h. The samples were collected using gel loading pipette tips, and 10-pl aliquots were quantitated after adding 3 ml of scintillation solution.

Kinetic Analysis-The standard kinase assay was performed for 5 min at 37 "C in the presence of 85 1.1~ ~-[l-~'c]pantothenate (specific activity 57.4 mCi/mmol), 250 ATP, 250 p MgCl,, 0.1 M Tris-HC1, pH 7.4, and protein in a final volume of 40 p1 as described by Vallari et al.

Pantothenate Kinase from E. coli 27053 (1987). Pantothenate kinase activity of wild-type (WT/WT) and heterodimer (WT/KlOlM) proteins was assayed a t 37 "C for 5 min a t the indicated concentrations of ATP/MgCl, (1:l) and pantothenate in the absence or the presence of the ATP analog (ATPyS). Kinetic constants were obtained from initial velocity measurements of 2 or 3 independent assays. Data were calculated as described by Gold et al. (1970). Because of the sigmoidal activity of wild-type pantothenate kinase as a function ofATP concentration, V,, was estimated from the initial velocity at 2.5 mM ATP, and the apparent K, values were calculated from Hill plots.

RESULTS Purification and Subunit Structure of Pantothenate

Kinase-To facilitate the purification of pantothenate kinase, the protein was overproduced using the dual plasmid expression system developed by Tabor (Tabor, 1990). The c o d gene was carried on plasmid pWS7-13-2 and was transcribed for 90 min following heat induction of T7 RNA polymerase carried on plasmid pGP1-2. The specific activity of pantothenate kinase in the total cell lysate of strain UB1005/pWS7-13-2/pGP1-2 following the 90-min induction period was 120 nmol/min/mg compared with 0.6 nmol/min/mg in strain UB1005. Pantothen- ate kinase was recovered in 71% yield in the 35-60% ammonium sulfate pellet.

Two methods were used to generate highly purified pantothenate kinase for kinetic characterization. The first method was affinity chromatography using Blue Sepharose (Fig. LA). Earlier work indicated that CoA binding to the protein was tightest a t pH 6.5 (Vallari et al., 1987), therefore the protein preparation was loaded onto a column equilibrated with a buffer at pH 6.5 to promote tight binding of the kinase. The column was washed with a buffer a t pH 7.5 containing 0.5 M NaCl to remove nonspecifically bound proteins, and then the enzyme was eluted with 0.25 mM CoA (Fig. lA). This purification method resulted in an average recovery of 17% and a final specific activity of 1.34 .c 0.1 pmoVmin/mg (number of experiments = 3). The pantothenate kinase preparation was nearly homogeneous as judged by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue (Fig. 1C). Two protein bands were observed with apparent molecular weights of 38 and 36 kDa corresponding to the predicted protein products of the c o d gene, 36.4 and 35.4 kDa, respectively. The c o d mRNA has two translational initiation sites, and the faster migrating protein is 8 amino acids shorter at the amino terminus than the slower migrating protein (Song and Jack- owski, 1992).

The second purification method was immunoaffinity chromatography (Fig. 1B). Pantothenate kinase purified by Blue Sepharose chromatography was used as an antigen to raise polyclonal antibodies in rabbits. Purified pantothenate kinase was coupled to Mi-Gel 10, and the resulting pantothenate kinase affinity column was used to purify monospecific antibodies (see "Experimental Procedures"). This monospecific antibody preparation was then in turn coupled to MI-Gel Hz and used as an immunoaffinity column to purify pantothenate kinase (Fig. 1B). This method also yielded nearly homogeneous pantothenate kinase as determined by SDS-polyacrylamide gel electrophoresis (Fig. 10. Pantothenate kinase activity purified by immunoaffinity chromatography was recovered in 6% yield with a specific activity of 0.47 5 0.2 pmoVmin/mg (number of experiments = 3). The lower specific activities of the kinase purified by immunoaffinity chromatography compared with the Blue Sepharose affinity chromatography were attributed to the high pH (11.5) required to elute the protein from the antibody matrix because prolonged exposure to pH 11.5 irreversibly inactivated the kinase. However, we did not detect a difference in the K,,, values for substrates or inhibitors of the enzyme prepared by the two different procedures. The immunoaffinity column was essential for the purification of mutant pantothenate

A. Blue Sepharose column

6oo00

4oo00

zoo00

0

1.5 0 0 2.

1.0 0

m

2 5

2 0.5 2

0 - 0.0

0 10 20 30 40 50 60 Fraction Number

B. Antibody column

Fraction M e r

C. Gel electrophoresis

1 2 3 4

FIG. 1. Purification of pantothenate kinase. First, pantothenate kinase was overproduced using the T7 expression system in strain vSlOO5/pWS7-13-2/pGP1-2; cell extracts were prepared, fractionated with ammonium sulfate, and dialyzed. Panel A, isolation of pantothenate kinase by Blue Sepharose chromatography. The 11.5-ml column was loaded with the dialyzed ammonium sulfate fraction and eluted with the indicated buffers. Pantothenate kinase was recovered by elution with 0.25 mM CoA. Panel B, purification of pantothenate kinase using immunoafinity chromatography. The total protein extract was loaded onto a 2-ml immunoaffinity column and eluted with the indicated buffers. Pantothenate kinase was eluted with 0.1 M triethylamine (TEA), pH 11.5, and collected into tubes containing 0.5 ml of 1 M "is-HC1, pH 8.0, to immediately neutralize the triethylamine. Panel C , SDS-polyacrylamide gel electrophoresis of purified pantothenate kinase. Protein samples from the crude cell extract (lane I), the ammonium sulfate precipitation step (lane 2 ) , the CoA eluate from Blue Sepharose chromatography (lane 31, and the triethylamine eluate from immunoafinity chromatography (lane 4 ). See "Experimental Procedures" for details of these methods.

kinase, which did not elute with GOA from the Blue Sepharose column.

Two methods were used to estimate the native subunit structure of pantothenate kinase (Fig. 2). Gel filtration chromatography indicated that the molecular weight of the kinase was 69 kDa using a column calibrated with typical globular proteins (Fig. 2A) . Sedimentation velocity experiments were used to


Gel filtration

200

100

10

1 S O 1.75 2.00 2.25

VelVo

B. Sedimentation velocity

10 ’ I I I I

25 30 35 40 45 50 - bottom Fraction Number top - FIG. 2. Subunit structure of pantothenate kinase. Panel A, pu-

rified pantothenate kinase (0) was fractionated by gel filtration chromatography using a Superose-12 column calibrated with standard pro-

velocity. Purified pantothenate kinase (0) and standard proteins (0) teins (0). Panel B , analysis of pantothenate kinase by sedimentation

were separated by sucrose density sedimentation. Abbreviations are as follows: ADH, alcohol dehydrogenase; BSA, bovine serum albumin; PK, pantothenate kinase; and OA, ovalbumin. See “Experimental Proce- dures’’ for details of these experiments.

verify the molecular weight determined by gel filtration chromatography. The position of pantothenate kinase in the sucrose gradient compared with the position of standard proteins indicated a molecular weight of 65 kDa (Fig. 2B). Based on these two results, we concluded that pantothenate kinase was a homodimer.

Kinetic Analysis of Pantothenate Kinase-The kinetic mechanism of pantothenate kinase was investigated by graphical analysis of initial velocity measurements at different substrate concentrations (Fig. 3). The intersecting lines obtained when the pantothenate concentration was varied at different fixed concentrations ofATP were consistent with a sequential mechanism (Fig. 3 A ) as opposed to a bireactant ping-pong mechanism that would yield a series of parallel lines. The analysis of the dependence of the reaction on ATP concentration at different pantothenate concentrations was more complex (Fig. 3B). The family of lines was not linear. One interpretation of this kinetic behavior was that the binding of ATP to the pantothenate kinase dimer exhibited positive cooperativity. Analysis of the data using a Hill plot was consistent with the interpretation that ATP binding to the kinase was highly cooperative with a Hill coefficient of 1.46 (Fig. 4). The nonlinear double-reciprocal plots attributed to cooperative ATP binding precluded further kinetic analysis of the wild-type dimer in determining

A. 400 1

I””

-100 -50 0 50 100 150 200

llpantothenate [mM”]

B’ 400

8 300 E Pantothenate 5.3 pM C

r E 200 z P 100 >

0 0 10 20 30 40

1/ATP [rnM-l] FIG. 3. Kinetic analysis of wild-type pantothenate kinase. Panel

A, double-reciprocal plots of initial velocity uersus pantothenate concentration at different fixed concentrations of ATP. Panel B, double-reciprocal plots of initial velocity uersus ATP concentrations at different fixed concentrations of pantothenate. Pantothenate kinase assays were performed as described under “Experimental Procedures.”

2 I I P Homodimer

WTIWT

Heterodimer WTlKlOlM l l ~ = 0.98

-1 I I I

1.0 2.0 3.0 4.0

and wild-typeKlOlM mutant heterodimers. Hill plots calculated FIG. 4. Cooperative binding of ATP in wild-type homodimers

from initial velocity measurements on purified wild-type homodimers (WT/WT, 0) as in Fig. 3 or wild-type/KlOlM mutant heterodimers (WT/ KlOlM, 0) as in Fig. 6. The kinase assays were performed at various ATP concentrations (32-2,500 pd at 85 p~ pantothenate.

the order of substrate binding in the postulated sequential mechanism.

Site-directed Mutagenesis of Pantothenate Kinase-Cata- lytically inactive pantothenate kinase was generated by mutagenesis of the predicted ATP binding site. Pantothenate

Pantothenate Kinase from E. coli 27055

kinase contains an A-type ATP binding consensus sequence, GXXAXGKS (Chin et al., 1988), located at residues 95-102 (Song and Jackowski, 1992). Previous analysis of A-type binding sites showed that the conserved lysine in this sequence is a critical residue required for ATP binding (Hannink and Donoghue, 1985; Snyder et at., 1985; Ebina et al., 1987). Site- directed mutagenesis was used to change lysine 101 to methionine (KlOlM) (see "Experimental Procedures"), and the mutant protein was expressed from plasmid pBS-KlOlM. The mutant gene did not complement the conditionally defective codl5(Ts) allele in strain DV73, although KlOlM-pantothenate kinase protein expression was 16-fold higher than wild-type levels as estimated from immunoblot analysis of cell extracts (not shown). Protein purXed by imm~oafflnity chromatography from cells expressing the KlOlM mutant possessed negli- gible kinase activity in vitro (<2 n m o ~ m i ~ m g ) . The molecular weight of KlOlM-pantothenate kinase was determined by gel filtration chromatography as described in Fig. 1, except immunoblot analysis of the fractions was used to locate the protein eluted from the column. These data indicated that K101M- pantothenate kinase had the same Stokes radius as the wild- type enzyme, illustrating that dimer formation was not affected by the mutation (not shown). These data demonstrate that Lys-101 was essential for pantothenate kinase activity, but the mutation of this residue to methionine did not interfere with dimer formation.

Eq~ili&rium Dialysis-ATP binding to wild-type and mutant pantothenate kinase was evaluated directly by equilibrium dialysis using the nonhydrolyzable ATP analog, [35SIATPyS. The dissociation constant (K,) was 2.1 5 0.1 p~ at 25 "C, and the number of binding sites calculated from Scatchard analysis was 1.9 2 O.l/mol of pantothenate kinase dimer, indicating that each subunit has a single ATP binding site. The kinetic Ki for ATPyS (3.5 PM at 25 "C) was similar to the Kd determined from equilibrium dialysis. The KlOlM mutant protein, purified by i ~ u n o a f f i ~ t y chromatography, did not bind [35S]ATPyS. CoA was previously shown to be a competitive inhibitor of pantothenate kinase with respect to ATP, suggesting that the ATP substrate and the CoA inhibitor bound to the same site on the enzyme (Vallari et al., 1987). We used equilibrium dialysis with wild-type and mutant proteins to further test this hypothesis. L3H1CoA bound to the wild-type kinase with a calculated K, of 6.7 5 1.4 p ~ , and the average number of CoA binding sites calculated from these experiments was 1.7 2 0.2/pantothenate kinase dimer. The K, for CoA measured from kinetic determinations (24 2 5 PM at 25 "C) was higher than the equilibrium dialysis measurement. One possible reason for the disparity is that the CoA Ki, which was calculated according to Cleland's equation for competitive inhibition (Cleland, 1963), is dependent on the ATP concentration and, as such, is not an intrinsic Ki and cannot be compared with the value of Kd. In addition, the pantothenate kinase protein concentration in the equilibrium dialysis experiments (0.5 p ~ ) was very high compared with the concentration used for the kinetic studies. Alternatively, variation between the K, values could be due to a small variation in the radiochemical specific activity determination for the [3HICoA prepared in our laboratory. Nevertheless, (3H]CoA binding to the KlOlM mutant was not detected. These data support the concept that ATP and CoA bind to the same site on the enzyme and that Lys-101 plays an important role in the binding of both ligands to the protein.

Expression of Wild-type and KlOlM Mutant Heterodimers- The technique of intragenic complementation was used to generate wild-type/KlOlM mutant heterodimers of pantothenate kinase. The KlOlM mutant cloned into the high copy number plasmid, pBS(+), and expressed in wild-type E. coli resulted in

TABLE I A comparison of the kinetic properties of wild-type homodimers

(WTi WT, and wild-type-KIOlM mutant heterodimers (WTlKlOIMl

Strain UB1005 UB10051 BS-KlOlM ( ~ ~ ) ~~~~0~~~

Total cell extract Relative pantothenate x 1 x175 Kinase levelu V,, (nmol/min/mg) 0.86 & 0.15 0.76 f 0.08

Specific activity (nmol/min/mg) 470 * 200 28 f 1 Pantothenate K,,, ( p a l b 36 * 4 41 f 7 ATP K,,, (wM)c 136 2 15 314 * 24

Purified pantothenate kinase

Protein levels were measured by immunoblotting using a standard curve prepared with purified pantothenate kinase to verify that the measurements were in the linear range.

Km values were determined at 125 PM ATP. Km values were determined from the Hill plots in Fig. 4.

a 17.5-fold increase in the level of pantothenate kinase protein recovered in crude cell extracts based on immunoblotting (Table I). The 17:1 ratio of mutant to wild-type protein sug- gested that almost all of the wild-type subunits would be com- plexed with KlOlM mutant subunits. The total activity in the two cell extracts was essentially the same, indicating that expression of the mutant pantothenate kinase subunits did not inhibit the enzyme. Immunoaffinity chromatography was used to purify pantothenate kinase from both strains, and the specific activity of the purified preparations was determined. Due to the presence of large amounts of inactive protein subunits in the extract, the specific activity of purified pantothenate kinase from strain UB1005/pBS-K101M was 16.5 times less than the specific activity of pantothenate kinase purified from strain UB1005 (Table I). The apparent K,,, for pantothenate was un- changed, but the apparent K,,, for ATP increased from 136 to 314 p~ (Table I). CoAinhibited the activity of wild-typeKlO1M mutant heterodimers. Thus, coexpression of KlOlM mutant subunits did not alter the total activity of pantothenate kinase in the extract, but there was a significant effect on the apparent K, for ATP.

Verification of ~eterodimeF Formation-Since KlOlM mutant subunits form dimers, it was anticipated that when they were coexpressed with wild-type protein, heterodimers between mutant and wild-type subunits would form. To verify this point, a derivative of plasmid PET-lSb, PET-KlOlM, was constructed (see "Experimental Procedures") to demonstrate the mixing of active and inactive subunits in vivo. Plasmid PET-KlOlM encoded a 332-amino acid protein with a predicted molecular weight of 38 kDa. The first 25 amino acids (MGSSH- HHHHHSSGL~RGSHMLGDP) were derived from the vector, and the 6 His residues (HisTag) allowed purification of the fusion protein by metal chelation chromato~aphy using a Ni"- charged resin. The 26th amino acid of the HisTag-KlOlM protein was encoded by the coaA gene and corresponded to amino acid 10 (Pro) of pantothenate kinase (Song and Jackowski, 1992). Expression of the HisTag-KlOlM protein was induced with 0.4 mM isopropyl-1-thio-p-D-galactopyranoside for 4 h, and the extracts were analyzed for the association of pantothenate kinase activity with Ni2+-charged affinity resin (Fig. 5). We reasoned that the dimerization of wild-type subunits with HisTag-KlOlM protein would result in the association of pantothenate kinase activity with the resin. In a control experiment, wild-type homodimer pantothenate kinase was exposed to the resin, and immunoblotting showed that wild-type kinase did not associate with the resin. Measurement of wild-type kinase activity in the same experiment showed that 9% of the kinase bound to the resin and 89% of the kinase was recovered in the unbound fraction. These data corroborate the immunoblot experiments and indicate that nonspecific binding to the


KDa

1 2 3 4 5 6 7 FIG. 5. Biochemical evidence for the formation of wild-type/

KlOlM heterodimers. Cell extracts (750 pg) were mixed with 100 pl of Ni*+-charged affinity resin to measure the binding of wild-type pantothenate kinase and HisTag-KlO1M mutant pantothenate kinase binding by activity measuremcnts and immunoblotting. Wild-type kinase exists in 38- and 36-kDa forms, and the HisTag-KlOlM mutant had an apparent molecular weight of 42 kDa on SDS-polyacrylamide gel electrophoresis due to the HisTag amino-terminal extension. Lune 1, purified pantothenate kinase ( C o d ) ; lune 2 , total cell extract from wild-type cells; lune 3, unbound fraction of the wild-type extract; lune 4, wild-type extract bound to the resin; lune 5, extract from wild-type cells expressing the HisTag-KlOlM mutant (Wild-type + his-KlOlM); lune 6, unbound fraction from the HisTag-KlOlM extract; and lune 7, bound fraction from the HisTag-KlO1M extract.

column was about 10%. In contrast, the HisTag-KlOlM bound to the column as indicated by immunoblotting (Fig. 5). The HisTag-KlOlM protein had an apparent molecular weight of 42 kDa based on SDS-polyacrylamide gel electrophoresis. The presence of a low level of HisTag-KlOlM protein in the unbound fraction indicated that the column did not bind all of the HisTag-protein in the cell extract. Due to the large excess of HisTag-KlOlM protein in the cell extract compared with wild- type protein, wild-type protein was not detected in the fractions by immunoblotting. In the presence of HisTag-KlOlM expression, 81% of the total pantothenate kinase activity bound to the resin, and 19% remained unbound. These data verify that when KlOlM mutant subunits were coexpressed with wild-type subunits, the two proteins formed heterodimers.

Kinetic Analysis of Wild-typeIKlO1M Mutant Hetero- dimers--Two key issues were resolved by the kinetic analysis of wild-type/KlOlM heterodimers. First, the double-reciprocal plots of initial velocity versus either pantothenate concentrations a t different fixed ATP concentrations or ATP concentrations at different fixed pantothenate concentrations were linear with no indication of cooperative binding of ATP (Fig. 6). This conclusion was confirmed with a Hill plot that showed that the wild-type/KlOlM heterodimers had a Hill coefficient of 0.98 (Fig. 4). The linearity of the double-reciprocal plots for both pantothenate and ATP permitted a kinetic analysis of pantothenate kinase and showed a pattern of intersecting lines that indicated that the reaction occurred by a sequential mechanism. Implicit in all sequential mechanisms is the assumption that the substrates must be present simultaneously at the enzyme’s active site before product formation can occur. Modifi- cation of the sequential mechanism as either random or ordered was differentiated using a competitive substrate inhibitor, and the order of addition of substrates was also determined by studying the pattern of inhibition in the presence of 100 ATPyS (Fig. 71, a nonreactive substrate analog. Non- competitive inhibition was observed when pantothenate was

A.

Ooo 600 -

F

2 200

.G 400 E c

> . 7

0

VPantothenate [rn”’]

B. 000 I I

600 - h

F

g 200 -

.C 400 E

- r

\ r >

0

t I - 2 0 0 ~ ” ” ” ‘ ” ’ ” ” ” ’ ’ ~ -10 -5 0 5 10

l/ATP [rnM-’] FIG. 6. Kinetic analysis of wild-type/KlOIM mutant het-

erodimers. Panel A, double-reciprocal plots of initial velocity versus pantothenate concentration at different fixed concentrations of ATP. Panel B , double-reciprocal plots of initial velocity versus ATP concentration at different fixed concentrations of pantothenate. Pantothenate kinase assays were performed as described under “Experimental Procedures.”

the variable substrate, and competitive inhibition was found when ATP was the variable substrate. These data were consistent with the conclusion that the pantothenate kinase reaction proceeded by a sequential mechanism with ATP binding first followed by pantothenate. Further experiments investigating the effects of inhibition by ADP, a product of the reaction, were carried out to verify the order of substrate addition and to examine the order of release of product. The ADP product was a competitive inhibitor with respect to ATP and noncompetitive with pantothenate (Fig. 8), which was consistent with either an Ordered Bi Bi, an Ordered Theorell-Chance Bi Bi mechanism, or rapid equilibrium with E-pantothenate-ADP as the dead-end complex. In these mechanisms, the nucleotide is the first substrate to add and the last to dissociate from the enzyme.

DISCUSSION

Our data provide a consistent picture of the subunit structure and kinetic mechanism of pantothenate kinase from E. coli. We developed two methods to prepare essentially homogeneous pantothenate kinase (Fig. 1) to verify and extend obser- vations made on the properties of the protein in crude cell extracts or partially purified preparations. The enzyme is a homodimer as evidenced from gel filtration chromatography and sedimentation velocity (Fig. 2). There is a single nucleotide binding site on each monomer, and ATP binding to the homodimer is highly cooperative (Fig. 4). CoA and its thioesters are the major regulators of pantothenate kinase activity and act by competitively inhibiting the binding ofATP to the kinase

Pantothenate Kinase from E. coli 27057

A. 800 I I I

600

400

200

0

-200 -100 -50 0 50 100 150

1lPantothenate [rnM-']

- I E" 1000 .r t E

100 pM ATPyS /p T I

0 ' I 1 I I

0 2 4 6 8 10

llATP [rnM"] FIG. 7. Substrate analog inhibition of wild-typeK101M het-

erodimers. Panel A, double-reciprocal plots of initial velocity versus

ATPyS. Panel 3, double-recip~cal plots of initial velocity uersus ATP pan~thenate concentration at 500 ~MATP in the presence or absence of

concentration at 40 p pantothenate in the presence or absence of ATPyS. Pantothenate kinase assays were performed as described under "Experimental Procedures."

(Vallari et al., 1987). Mutagenesis of a critical lysine at position 101 abolished both ATP and CoA binding, corroborating the conclusion from kinetic analysis that these two nucleotides bind to the same site on the monomer. Considering the struc- tural similarity between ATP and CoA and the uncompetitive inhibition pattern of CoA with respect to pantothenate, it is likely that the ADP moiety of CoA determines the interaction between protein and ligand. However, CoA binding did not exhibit cooperative behavior. Overproduction of wild-type pantothenate kinase did not propo~ionally increase the intracellular CoAcontent, even in the presence of abundant pantothenate (Song and Jackowski, 1992), which points to the inhibition of the kinase by CoA as the primary mechanism regulating the intracellular pool of CoA. Based on our work, feedback regulation of pantothenate kinase involves the competitive binding of GOA to the ATP site, which blocks ATP binding at one site and prevents positive cooperative ATP binding to the second site on the dimer.

Cooperati~ty between protein subunits is not unusual, but kinetic analysis of the nature of the biochemical interaction between the substrates and pantothenate kinase was compli- cated by subunit interactions that generated nonlinear kinetic plots. The formation of chimeric dimers consisting of one wild- type protein subunit together with a subunit containing the KlOlM mutation with a defective ATP binding site was achieved by coexpression of the two COCA alleles in vivo. The single copy wild-type subunit was encoded by the chromosomal C O C A allele in strain UB1005, and approximately 17 copies of

A. I

lipantothenate [rn"']

B. 1000 1 ll.".-

I

-2 0 2 4 6 a 10

1IATP [rnM"] FIG. 8. Product inhibition of wild-typeKlO1M heterodimers.

Punel A, double-reciprocal plots of initial velocity versus pantothenate concentration at 500 p~ ATP in the presence or absence of ADP. PuneE B, double-reciprocal plots of initial velocity uersus ATP concentration at 40 p p a n ~ t h e n a ~ in the presence or absence of ADP. Pantothenate kinase assays were performed as described under "Experimental Procedures."

the mutant pantothenate kinase were expressed from the c d ( K l 0 l M ) allele encoded by plasmid pBS-KlOlM. Thus, the mixture contained primarily enzymatically inactive mutated dimers ( K l O l ~ l O l M ) and enzymatically active chimeric dimers (WTKlOlM). A significant amount of WTNVT dimers was unlikely due to the substantially lower number of wild- type molecules in the cell. This point is supported by the shift in the Hill coefficient from nH = 1.46 for wild-type protein dimers to nH = 0.96 for the enzyme preparation containing WTKlOlM chimeric dimers (Fig. 4). Elimination of the second colla~rating ATP site on pan~thenate kinase by genetic mutation of Lys-101 resulted in simplification of the kinetic analysis and led to the conclusion that the phosphorylation reaction catalyzed by pantothenate kinase proceeded by a sequential mechanism with ATP binding first followed by pantothenate a t a different site on the protein.

More information is required to determine if similar mechanisms control pantothenate kinases from other organisms. En- zymes from mammals and plants have not been extensively purified or subjected to a thorough kinetic analysis; however, the available data indicate that there are signficant similari- ties to the E. coli kinase. It is clear that CoA and its thioesters are potent inhibitors of pantothenate kinase from a variety of mammalian sources (Abiko et al., 1972; Karasawa et al., 1972; Halvorsen and Skrede, 1982; Fisher et al., 1985; Fisher and Neely, 1985). Pantothenate kinase from plants is somewhat different since this enzyme is inhibited by malonyl-CoA but not by CoA or acetyl-coA (Falk and Guerra, 1993). In mammais, CoA inhibition is known to be noncompetitive with respect to


pantothenate, but it is not known whether CoA inhibition is competitive with respect to ATP. Also it remains to be determined whether ATP exhibits cooperative binding, if CoA inhib- its activity by association with the ATP site, or if the kinases from other organisms possess a dimer subunit structure. The cooperative interactions between subunits and the competitive inhibition of ATP binding to CoA described in this report provide an enzymatic basis for the understanding of the regulation of cellular CoA content and suggests that determining whether these mechanisms also operate in eukaryotic systems will be the next important step in understanding the relationship between pantothenate kinase activity, CoA concentration, and energy metabolism in mammalian cells.

Acknowledgment-We thank Chuck Rock for helpful discussions.

REFERENCES

Abiko, Y. (1975) in Metabolic Pathways (Greenburg, D. M., ed) pp. 1-25, Academic

Abiko, Y., Ashida, S., and Shimizu, M. (1972) Biochim. Biophys. Acta 268,364372 Barbarat, B., and Podevin, R. A. (1986) J. Biol. Chem. 261, 14455-14460 Booth, B. R. (1980) Biochem. Biophys. Res. Commun. 94,1029-1036 Bradford, M. M. (1976) Anal. Biochem. 72,248-254 Chin, D. T., Gaff, S. A,, Webster, T., Smith, T., and Goldberg, A. L. (1988) J. Biol.

Cleland, W. W. (1963) Biochim. Biophys. Acta 67,173-187 Dawes, E. A., and Large, P. J. (1982) in Biochemistry ofBacteria1 Growth (Man-

delstam. J.. McQuillen. K.. and Dawes, I., eds) PP. 126-158. John Wilev & Sons,

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Chem. 263, 11718-11728

Inc., New York Ebina, Y., A r a k i , E., Taira, M., Shimada, F., Mori, M., Craik, C. S., Siddle, K.,

Pierce, S. B., Roth, R. A,, and Rutter, W. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,704-708

. . . . . "

Falk, K. L., and Guerra, D. J. (1993) Arch. Biochem. Biophys. 301,424-430

Fisher, M. N., Robishaw, J. D., and Neely, J. R. (1985) J. Biol. Chem. 260,15745- Fisher, M. N., and Neely, J . R. (1985) FEES Lett. 190,293-296

Gold,A. M., Johnson, R. M., and Tseng, J. K. (1970) J. Biol. Chem. 246,2564-2572

Hannink, M., and Donoghue, D. J. (1985) Proc. Natl. Acad. Sci. U. 5. A. 82,7894- Halvorsen, O., and Skrede, S. (1982) Eur. J. Biochem. 124, 211-215

7998 Harlow, E., and Lane, D. (1988) inAntibodies:A Laboratory Manual, pp. 519-552,

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Jackowski, S., and Alix, J.-H. (1990) J. Bacteriol. 172, 38424848 Jackowski, S., and Rock, C. 0. (1981) J. Bacteriol. 148,926-932 Jackowski, S., and Rock, C. 0. (1986) J. Bacteriol. 166, 866-871 Karasawa, T.,Yoshida, K., Furukawa, K., and Hosiki, K. (197215. Biochem. (Tokyo)

Lopaschuk, G. D., Michalak, M., Tsang, H. (1987) J. Biol. Chem. 262, 3615-3619 Magnuson, K., Jackowski, S., Rock, C. O., and Cronan, J. E., Jr. (1993) Microbiol.

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Miller, J. H. (1972) in Experiments in Molecular Genetics. pp. 433, Cold Spring Reu. 57,522-542

Perkins, S. J. (1986) Eur. J. Biochem. 157, 169-180 Harbor Laboratory, Cold Spring Harbor, NY

Robishaw, J. D., and Neely, J. R. (1984) Am. J. Physiol. 246, H532-H541 Robishaw, J. D., and Neely, J. R. (1985) Am. J. Physiol. 248, El-E9 Robishaw, J. D., Berkich, D., and Neely, J. R. (1982) J. Biol. Chem. 267, 10967-

Snyder, M. A,, Bishop, J. M., McGrath, J. P., and Levinson, A. D. (1985) Mol. Cell.

Song, W.J., and Jackowski, S. (1992) J. Bacteriol. 174, 6411-6417 Tabor, S. (1990) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent,

R., Kington, R., Moore, D. D., Seidman, J . G., Smith, J. A,, and Struhl, K., eds) pp.16.2.1-16.2.11, Greene Publishing and Wiley-Interscience, New York

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Vallari, D. S., and Jackowski, S. (1988) J. Bacteriol. 170, 39614966 Vallari, D. S., and Rock, C. 0. (1985a) J. Bacteriol. 164, 136142 Vallari, D. S., and Rock, C. 0. (1985b) J. Bacteriol. 162, 11561161 Vallari, D. S., and Rock, C. 0. (1987) J. Bacteriol. 169, 5795-5800 Vallari, D. S., Jackowski, S., and Rock, C. 0. (1987) J. Biol. Chem. 262,

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tiie journal of biological chemistry 269. no. 43, 28 ... · water, 5 ml of binding buffer, and 3 ml...

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