characterization of the atp binding site on escherichia coli dna

Vol. 265, No. 34, Issue of December 5, pp. 21342-21349, 1990 Printed in V. S. A.

Characterization of the ATP Binding Site on Escherichia coli DNA Gyrase AFFINITY LABELING OF Lys-103 AND Lys-110 OF THE B SUBUNIT BY PYRIDOXAL 5’-DIPHOSPHO-5’-ADENOSINE*

(Received for publication, June 22, 1990)

James K. Tamura$ and Martin Gellert From the Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes if Health, Bethesda,-Maryland 20892

We have labeled the adenosine triphosphate binding site of Escherichia coli DNA gyrase with the ATP affinity analog, [3H]pyridoxal 5’-diphospho-5’-adenosine (PLP-AMP). PLP-AMP strongly inhibits the ATP- ase and DNA supercoiling activities of DNA gyrase, with 50% inhibition occurring at 7.5 pM inhibitor. ATP and ADP compete with PLP-AMP for binding and protect the enzyme against inhibition. The labeling appears to proceed by a Schiff base complex between the I-formyl group of the pyridoxyl moiety of PLP- AMP and a protein primary amino group, since the inhibition and reagent labeling are reversible unless the complex is treated with NaBH4. Complete inactivation is estimated to occur upon the covalent incorporation of 2 mol of inhibitor/m01 of gyrase. The K,,, for ATP was found to be unchanged for partially inhibited enzyme samples, suggesting an all-or-none type of inhibition.

A 3H-labeled peptide spanning residues 93-131 of the B protein was isolated from a V-8 protease digest. Radioactive peaks corresponding to Lys-103 and Lys- 110 were found during the Edman degradation, suggesting that these amino acids form part of the ATP binding site. A comparison of the amino acid sequence in this region with the sequences of other type II topoisomerases indicates the possible location of a common ATP binding domain.

DNA topoisomerases are a class of enzymes which catalyze the interconversions between different topological isomers of DNA (for recent reviews see Vosberg, 1985; Maxwell and Gellert, 1986; Wang, 1985, 1987). The pathway for these reactions involves a transient breakage of one DNA strand (type I DNA topoisomerase), or a concerted breakage of both strands (type II DNA topoisomerase), followed by rejoining of the DNA backbone. With both types of enzymes, the DNA breakage and reunion-/reactions have been found to proceed via a covalent protein-DNA intermediate. As a consequence of breaking and rejoining the DNA backbone, different topoisomerases will catalyze a variety of reactions, including re- laxation and supercoiling, knotting and unknotting, and ca- tenation and decatenation of duplex circles.

Among type II topoisomerases, bacterial DNA gyrase is the

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

$ Associate of the National Research Council during the course of this work.

only enzyme capable of transforming relaxed closed circular DNA into a negatively supercoiled state, in a reaction coupled to ATP hydrolysis. The mechanism is thought to involve vectorial transport of a double stranded DNA segment through a transient double strand break in the direction that serves to decrease the linking number. The strands are then resealed to yield an underwound closed circular DNA molecule. In prokaryotes, DNA gyrase is an important element in many vital cellular functions, including DNA replication, genetic recombination, and the control of gene expression.

DNA gyrase contains two different subunits, designated A and B, of molecular weights 97,000 and 90,000, respectively (Swanberg and Wang, 1987; Yamagishi et al., 1986; Adachi et al., 1987), which are assembled to form an active A2Bz complex (Klevan and Wang, 1980). The enzyme binds a segment of DNA greater than 100 base pairs in length (Liu and Wang, 1978a; Fisher et al., 1981; Kirkegaard and Wang, 1981; Mor- rison and Cozzarelli, 1981; Maxwell and Gellert, 1984; Rau et al., 1987) wrapped around the protein in a positive superhel- ical sense (Liu and Wang, 1978b).

The B subunits carry the catalytic sites for ATP hydrolysis (Staudenbauer and Orr, 1981; Maxwell and Gellert, 1984). The ATPase activity of the isolated B subunit is low but is greatly stimulated in the presence of the A subunit and DNA; this stimulation is largely nonspecific with regard to DNA sequence (Mizuuchi et al., 1978; Sugino and Cozzarelli, 1980; Maxwell and Gellert, 1984). The nucleotide requirement, however, appears quite specific for ATP (Sugino and Cozzarelli, 1980).

The region on the primary sequence involved in ATP binding has remained elusive due to the absence of unambig- uous regions in gyrase that fit consensus sequences A and B, common to many ATP-utilizing enzymes (Walker et al., 1982; Mildvan and Fry, 1987; Serrano, 1988). In order to identify this region, we have utilized the ATP affinity analog, pyri- doxal5’-diphospho-5’-adenosine (PLP-AMP)l to specifically label the ATP binding site on gyrase. PLP-AMP has proven to be an effective structural analog of ATP for modifying lysyl residues at nucleotide binding sites on a wide variety of enzymes (Tamura et al., 1986,1988; Tagaya and Fukui, 1986; Tagaya et al., 1987; Rao et al., 1988; Dombroski et al., 1988; Miziorko et al., 1990). The reactive 4-formyl group on the pyridoxyl moiety forms a Schiff base complex with the t- amino groups of proximal lysines, and upon borohydride reduction yields a stable derivative. We report here the specific labeling of Lys-103 and Lys-110 of the B subunit by

1 The abbreviations used are: PLP-AMP, pyridoxal B’-diphospho- 5’-adenosine; HPLC, high performance liquid chromatography; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid.

21342

by guest on March 14, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

ATP Binding Site on DNA Gyrase

PLP-AMP. Evidence is presented suggesting that these lysyl residues are at the ATP binding site of gyrase.

EXPERIMENTAL PROCEDURES Escherichin coli DNA Gyrose-Gyrase A and B subunits were

isolated individually from the overproducing strains N4186 and MK47, respectively (Mizuuchi et al., 1984). When necessary, the subunits were concentrated in Centricon- microconcentrators (Amicon Corp., Danvers, MA) to 0.5-1.0 mg/ml. Aliquots of 100 ~1 were desalted on l-ml spin columns containing Sephadex G-50-80 (Penefsky, 1977) equilibrated with 50 mM Hepes-KOH (pH 7.5), 24 mM KCl, 10 mM potassium phosphate, 6 mM MgCl,, and 6.5% (w/v) glycerol (buffer 1). Protein concentrations were determined by the Bio-Rad protein assay using bovine serum albumin (Calbiochem) as standard. The concentration of bovine serum albumin solutions was determined by the absorbance at 280 nm using an extinction coefficient of 0.66 for an 0.1% solution (Tanford and Roberts, 1952). The specific activities of the A and B proteins determined in DNA supercoiling assays (defined according to Mizuuchi et al., 1984) were 8 x lo6 and 9 ~-10~ units/mg of protein, respectively. The specific ATPase activity of the A,B, comnlex usine linear nUC9 DNA sub- strate and 0.4 r& ATP in the st&dard assay (described below) was typically between 0.25 and 0.3 pmoles of ATP hydrolyzed/min/mg of protein.

Enzyme Assays-Two methods were used for measuring ATPase activity. The first method measured initial rates of 32Pi liberation from the hydrolysis of lr-32PlATP. The reaction mixtures contained buffer 2 (buffer-1 containing 1.8 mM spermidine and 5 mM dithiothreitol), plus 15 rg/ml linear pUC9 DNA and 0.4 mM [-y-32P]ATP. The reaction was initiated by addition of the enzyme-DNA complex and incubated at 25 “C. Aliquots were removed at various times during the initial phase of the reaction where the rate of ATP hvdrolvsis was linear, and assayed for 3zPi according to the method of Grubmeyer et al., (1982). The assays were performed directly in 3-ml plastic scintillation vials.

The second method measured the rate of absorbance decrease at 340 nm in an assay that coupled the hydrolysis of ATP to the oxidation of NADH. The reaction was performed at 25 “C in a total volume of 0.3 ml in the same reaction mixture as above for the 32Pi assay with the addition of 100 pg/ml pyruvate kinase and 20 rg/ml lactate dehydrogenase from 50% glycerol solutions (Boehringer Mannheim), 2 mM phosphoenolpyruvate, and NADH to give -an A340 nm of 0.3. The reaction was initiated bv the addition of the avrase- DNA complex (usually about 12 pmol) and the time course-of the absorbance decrease was followed using a Cary 210 spectrophotometer with a Brinkman Instruments RM3 temperature controller. The rate of ATP hydrolysis due to contaminating ATPases was found to be less than 2% of the total, based on ATPase measurements in the presence of 15 fiM novobiocin. At this concentration, novobiocin had no effect on the coupled enzyme assay. Both methods for assaying the ATPase activity gave similar results. However, the second method proved to be easier and gave more reliable results than the first because the hydrolysis product, ADP, was converted back to ATP by pyruvate kinase, thus allowing a constant rate of ATP hydrolysis to proceed over the entire course of the assay.

DNA supercoiling assays were performed as described by Mizuuchi et al. (1984). The reaction was initiated by the addition of gyrase and incubated for 60 min at 25 “C. The amount of linear pUC9 DNA added with the enzyme as carryover (see below) was less than 0.15% of the concentration of pBR322 DNA in the assay.

Modification of Gyrase by PLP-AMP-The A and B subunits were mixed in equimolar amounts (l-2.5 uM each) in buffer 1. The subunits were incubated for 30 min at room temperature in the presence of linear pUC9 DNA at 140 rre of DNA/nmol of svrase. which corresponds- to about 215 base pa&/enzyme molecule. An aliquot of [3H] PLP-AMP, synthesized according to Tamura et al. (1986), was dried in a Speed Vat concentrator and dissolved in a minimal volume of buffer 1. The concentration was determined from the absorbance at 388 nm in 0.1 N NaOH, using a molar extinction coefficient of 6600 M-’ cm-’ (Tamura et al., 1986). The gyrase-DNA complex was reacted with 13HlPLP-AMP in a total volume of 60 ul under low light conditions for 45 min at room temperature. A 5-pi aliquot of a freshly prepared solution of 130 mM NaBHl in 5 mM NaOH was added to the reaction mixture, giving a final NaBHl concentration of 10 mM. The mixture was incubated for 24 s, followed by the addition of 55 pl of 0.5 mg/ml bovine serum albumin in buffer 1. Aliquots of 100 ~1 were applied to l-ml spin columns equilibrated with buffer 2 contain-

ing 0.25 mg/ml bovine serum albumin to remove unbound inhibitor and excess NaBH,. The time between the addition of NaBH, and the application of samples to the spin columns was held constant at 35 s. This time was sufficient to completely reduce the Schiff base complex to a stable pyridoxamine derivative while still minimizing foaming. Longer times of exposure to NaBH, resulted in a decrease in the ATPase activity of controls.

The spin column effluents were collected directly in preweighed 1.5-ml Eppendorf tubes placed in the bottom of the test tubes holding the spin columns. The Eppendorf tubes were weighed again and the volumes of the spin column effluents were calculated from the net weights and the density of the solution. Aliquots of the modified enzyme were assayed for radioactivity, ATPase activity, and supercoiling activities as described. The concentrations of gyrase emerging from the spin columns were based on the recovery of ATPase activity of controls that lacked PLP-AMP. Under the above conditions. the recovery of ATPase activity was reproducibly found to be 87:90% and the nonspecific leak-through of unbound radioactivity was less than 1 part in 20,000.

Peptide Purification and Amino Acid Sequencing-Modified gyrase was prepared on a larger scale by incubating the gyrase-DNAcomplex with a concentration of [3H]PLP-AMP that resulted in about 0.9 mol of incorporated analog/mole of gyrase. The Schiff base complex was reduced for 2 min with freshly solubilized 5 mM NaBHI added from a stock solution in 10 mM NaOH. Aliquots were added to a series of l-ml spin columns previously equilibrated with 10 mM Hepes-KOH (pH 7.5) containing 0.2 mg/ml bovine serum albumin. The enzyme samples, containing approximately 0.5 mg of protein, were lyophilized and dissolved in 100 ~1 of 0.1 M NHIHCO, (pH 7.8), 2 mM EDTA, and 0.2% sodium dodecyl sulfate. Staphylococcus aureus V-8 protease (Pierce Chemical Co.) from a 5 mg/ml stock solution in HZ0 was added to give a final proteasegyrase ratio of 1:25 (w/w). After 12 h at 34 “C, an equal aliquot of the protease was added and the incubation was continued for an additional 2 h at 34 “C. The mixture was further incubated with 5 mM dithiothreitol for 1 h and then acidified with 0.5% trifluoroacetic acid to convert HCO; to CO2 gas before HPLC analysis. The sample was injected directly into a Beckman HPLC apparatus, and the peptides were resolved initially on a 4.6 x 250- mm Vydac 214-TP546 C, column containing 5 pm packing. All HPLC separations were performed at a flow rate of 0.5 ml/min. After 5 min of washing with 99% solvent A (0.1% trifluoroacetic acid in HzO) and 1% solvent B (0.1% trifluoroacetic acid in 99.9% acetonitrile), the peptides were eluted with a linear gradient developed from l-40% solvent B at 0.25% solvent B/min, followed by 1% solvent B/min from 40-100% solvent B. The eluate was collected in 0.5- or l-ml fractions, and aliquots were counted for radioactivity. The fractions containing the 3H-labeled peptide were pooled and lyophilized. The residue was dissolved in 100 ~1 of 40% solvent B and purified on a 4.6 X 250-mm Vydac (218-TP546) Cl8 column using a linear gradient developed from 20-40% solvent B at 0.15% solvent B/min.

Alternatively, the dried residue containing the 3H-labeled peptide from the Cq column was dissolved in 100 ~1 of 0.1 M Tris-HCl (pH 8), 0.1 M CaC12, 2 M urea, 50 mM methylamine, and digested with chymotrypsin at an approximate protease:peptide ratio of 1:12 (w/ w). After 3 h at room temperature an equal aliquot of chymotrypsin was added and the incubation continued for an additional 8 h. The chymotryptic digest was resolved on a C, column using a linear gradient from l-70% solvent B at 0.375% solvent B/min. Fractions from each peak containing radioactivity were pooled separately and then purified on a Cl* column. The gradients were developed from l- 30% solvent B at 0.2% solvent B/min.

The purified peptides were lyophilized, dissolved in 150 ~1 of 40% solvent B, and sequenced on an Applied Biosystems model 471A protein sequencer. The phenylthiohydantoin amino acid derivative released in each cycle of the Edman degradation was automatically transferred to an on-line Applied Biosystems model 130A separation unit, of which 50 ~1 was analyzed for amino acid identity on a 2.1 x 22-cm Cl8 column while the remainder was directed to a fraction collector for measurement of the radiactivity.

Polyacrylamide Gel Electrophoresis-Gyrase labeled by [3H]PLP- AMP was run on 1.5-mm-thick 7.5% acrylamide gels containing 0.375 M Tris-HCl (pH 8.8) and 0.1% sodium dodecyl sulfate according to Laemmli (1970) on a Bio-Rad Protean slab-gel apparatus. The protein bands were visualized with Coomassie Blue, sliced from the gel, and incubated in 1.8 ml of a 9:l mixture of NCS tissue solubilizer (Amer- sham Corp.): Hz0 at 50 “C for 2 h in tightly sealed glass scintillation vials. The mixture was acidified with acetic acid and counted for


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

ATP Binding Site on DNA Gym-se

radioactivity in order to assess the amount of covalent incorporation of [3H]PLP-AMP into the gyrase A and B subunits.

RESULTS

Inactivation of DNA Gyrase by PLP-AMP-The ATPase activity of DNA gyrase becomes activated in the presence of duplex DNA (Mizuuchi et al., 1978; Sugino et al., 1978). This activation is believed to become optimal when DNA binds to two sites on the enzyme and this requires a length of DNA greater than 100 base pairs (Maxwell and Gellert, 1984). Thus, gyrase was preincubated with linear pUC9 DNA@768 base pairs) at a ratio greater than 200 base pairs/gyrase molecule. Under these conditions, it is expected that all gyrase molecules are saturated with a single topological form of DNA, thereby minimizing enzyme heterogeneity during the reaction with PLP-AMP.

When the gyrase-DNA complex was incubated with PLP- AMP and then treated with NaBH4, the ATPase activity was strongly inhibited (Fig. 1). Inhibition by 50% was obtained at 7.5 pM PLP-AMP and more extensive inhibition was achieved at higher concentrations, reaching 91% at 48 pM inhibitor. The addition of NaBH., without PLP-AMP to a control enzyme resulted in only a minor decrease (5%) in the specific ATPase activity. If the enzyme was preincubated with PLP- AMP, but not treated with NaBH+ the ATPase activity measured immediately following the removal of unbound inhibitor on spin columns increased during the time course of the assay. In addition, the 3H label was readily lost from the protein pellet upon precipitation with trichloroacetic acid, unless the samples were treated with NaBH4. This behavior is consistent with the known reversibility of Schiff base complexes formed between the 4-formyl group of the pyridoxyl moiety and a protein primary amino group, which can be stabilized by borohydride reduction.

The ability of ADP to compete with PLP-AMP for binding is shown in Fig. 2. ADP effectively protected against inhibition by PLP-AMP, and the degree of protection increased with increasing concentration of ADP. The extent of covalent modification was also progressively decreased at higher ADP concentration (data not shown). A similar protective effect was afforded by ATP, although interpretation of the results is complicated by the hydrolysis of ATP to ADP and inorganic phosphate. These results imply that PLP-AMP attaches to the nucleotide binding site, and that its binding is mutually

04 , 1 , 1 0 10 20 30 40 50

[PLP-AMP]. PM

FIG. 1. Inactivation of DNA gyrase by PLP-AMP. Gyrase at 1 pM in the presence of 140 pg/ml pUC9 DNA was incubated with varying concentrations of [3H]PLP-AMP in buffer 1 for 45 min. The samples were treated with NaBH,, passed through spin columns, and assayed as described under “Experimental Procedures.” The results are expressed as percentage ATPase activity of control enzyme that was not incubated with PLP-AMP but otherwise subjected to the same treatment. The concentration of free inhibitor is plotted on the abscissa.

FIG. 2. Effect of ADP on the inactivation of DNA gyrase by PLP-AMP. The conditions were the same as described in the Zegend to Fig. 1 except that the enzyme-DNA complex was incubated with the indicated concentrations of ADP for 30 min prior to the addition of 0 (O), 10 (O), 20 (O), or 30 (m) pM PLP-AMP.

0 0.0 0.5 1.0 1.5 2.0

PLP-AMP/Gyrase (mol/mol)

FIG. 3. Stoichiometry of [3H]PLP-AMP binding. The experiment was essentially the same as described in the legend to Fig. 1 but the percentage of residual ATPase activity is plotted as a function of moles of PLP-AMP incorporated per mol of gyrase. The initial points corresponding to low levels of incorporated label were obtained from samples containing 1.5 times higher gyrase concentration in order to more accurately quantitate the amount of [3H]PLP-AMP bound to the enzyme. The solid line shows the anticipated effect for full inactivation of the ATPase activity resulting from binding to a single catalytic site on gyrase. The details of the analysis are described under “Results.”

exclusive to that of ATP or ADP, by virtue of its structural similarity.

How specific is the covalent modification by PLP-AMP? It can be seen in Fig. 3 that there is a nonlinear correlation between the loss of ATPase activity and PLP-AMP binding over the entire range of the reaction. Extrapolation from the later region of the curve to 100% inhibition of ATPase activity corresponds to binding approximately 2 mol of inhibitor/m01 of gyrase. On the other hand, a similar extrapolation from the initial stages of the curve suggested that the incorporation of only 1 mol of PLP-AMP/mol of gyrase was sufficient to inhibit the ATPase activity. We do not believe that the biphasic profile is due to differential binding to a fraction of enzyme not complexed with DNA since our reactions contained more than 200 base pairs DNA/gyrase molecule; above 150 base pairs/gyrase, the stoichiometry of PLP-AMP binding was independent of DNA concentration and the DNA- stimulated ATPase activity was maximal. Thus, inhibition appears to result from less than stoichiometric binding to the ATP binding sites.

The inhibition of ATPase activity by PLP-AMP modification appears to parallel the loss of DNA supercoiling activity. Fig. 4 shows that enzyme inhibited so as to retain 51% (Fig. 4B) and 21% (Fig. 4C) of the original ATPase activity re- quired about two and four times, respectively, the quantity of the control enzyme (Fig. 4A) to yield 1 unit of DNA super-


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

ATP Binding Site on DNA Gyrczse 21345

FIG. 4. Inhibition of DNA AMP. DNA gyrase was incubate

mtivity by PLP- P at concentrations

sufficient to give 51 and 21% of the original ATPase activity of the control, respectively. These samples contained 0.62 and 1.3 mol of inhibitor/m01 of gyrase, respectively. The control enzyme was treated similarly but without the addition of PLP-AMP. Serial dilutions of the eluates from the spin columns were made into 50 mM Tris-HCI (DH 7.5). 0.1 M KCI. 0.2 mM EDTA. 5 mM dithiothreitol. 50% elvcerol.

- ”

and 2 mg/ml bovine serum albumin. The DNA supercoiling reactions were initiated by the addition of 1 ~1 of diluted gyrase into the assay mixture (total volume, 70 rl). Samples were later electrophoresed in 0.8% agarose gels as described under “Experimental Procedures.” A, control; B, gyrase with 51% of the original ATPase activity; C, gyrase with 21% of the original ATPase activity. The amounts of gyrase (in nanograms) added to the assay mixtures are shown in lanes I-4: lane 1, 5.47; lane 2, 2.74; lane 3, 1.37; lane 4, 0.68. The bands corresponding to relaxed (Rel) and supercoiled (SC) DNA are indicated. The Rel band consists primarily of closed circular DNA plus minor amounts of nicked DNA. One unit of supercoiling activity is defined as con- verting half of the relaxed closed circular pBR322 DNA to a fully supercoiled form (Mizuuchi et al., 1984).

l/[ATP]. mM-’

FIG. 5. Kinetic analysis of the inhibitory effect of PLP- AMP on DNA gyrase. The ATPase activity of partially inhibited gyrase samples was measured in the presence of varying concentrations of MgATP. Enzyme samples that had been incubated with 0 (0), 5 (O), 9 (III), or 15 (m) pM PLP-AMP were treated with NaBHa, applied to spin columns, and assayed for ATPase activity as described under “Experimental Procedures.” The stoichiometry of incorporated PLP-AMP was respectively 0, 0.51, 0.86, and 1.1 mol/mol of gyrase. The K,,, values for ATP determined from linear regression analysis were 0.30, 0.26, 0.27, and 0.29 mM, respectively.

coiling activity (see Fig. 4: A, lane 3; B, lane 2; and C, lane 1). For measurements of the ATPase activity of modified gyr-

ase samples, we used 0.4 mM ATP. The possibility remained that PLP-AMP reacted with lysyl residues distant from the nucleotide binding site and induced a protein conformational change that lowered the affinity for ATP. However, Fig. 5 shows that the K,,, for ATP of enzyme samples containing between 0.51 and 1.1 mol of inhibitor/m01 of gyrase ranged between 0.26 and 0.29 mM, essentially the same as the unmodified control (0.30 mM). Only the V,,, for ATP hydrolysis changed after modification. This suggests an all-or-none inhibitory effect, typical of an inactivation process. Thus, the residual ATPase activity observed with partially inactivated enzyme is probably not due to modified enzyme with altered kinetic properties.

Identification of the Subunit Modified by PLP-AMP-Gyr- ase samples were incubated with different concentrations of PLP-AMP, reduced with [3H]NaBH4, and the A and B subunits were resolved on denaturing 7.5% polyacrylamide gels. The subunits were visualized by Coomassie Blue staining and sliced from the gel for radioactive scintillation counting. In three samples labeled to differing extents, 86-90% of the radioactivity was associated with the B subunit (Table I). The specificity of modification of the B subunit decreased only slightly in the sample with the highest amount of incorporated analog. However, when 2 mM ADP was included during the incubation with PLP-AMP (sample 4), the stoichiometry of PLP-AMP binding decreased by about 1 mol/mol of gyrase, and this decrease can be almost exclusively attributed to a diminished incorporation into the B subunit (from 1.44 to 0.46 mol/mol of B dimer). The results are consistent with PLP-AMP binding to the nucleotide binding site on the B subunit.

The result showing the stoichiometry of PLP-AMP binding versus the remaining ATPase activity (see Fig. 3) was thus analyzed based on the assumptions that total inactivation of the tetramer results from PLP-AMP binding to only one of two catalytic sites, that the 10% residual analog bound to the A subunits does not inhibit the ATPase activity, that binding to either site is equally probable, and that binding to one site does not affect subsequent binding to the other. At varying values of PLP-AMP bound/gyrase, the fractions of occupied (X) and unoccupied (Y) sites were calculated. Thus, the fraction of enzyme containing 0, 1, and 2 bound molecules of PLP-AMP are represented by (Y)“, 2(XY), and (X)‘, respectively. The fractional ATPase activity is equal to ( Y)2 because gyrase without bound analog is assumed to be the only active species. The computer-generated theoretical curve gives a reasonable fit to the experimental data points.

Purification and Sequence Determination of a Peptide Con- taining Covalently Bound PLP-AMP-Preparative amounts of gyrase (0.5 mg), containing 0.9 mol of inhibitor/m01 of enzyme, were digested exhaustively with V-8 protease and the peptides were separated as described under “Experimental Procedures.” Fig. 6 shows the elution of peptides from the digest as monitored by their absorbance at 220 nm as well as radioactivity. From the known amino acid sequences of the A and B subunits, there should be a total of 150 peptides generated from V-8 proteolysis, assuming complete cleavage after all glutamic acid residues (Drapeau, 1977). A single major radioactive peak eluted in fractions 115-118 (32% solvent B); these fractions were pooled and further purified by a second chromatography on a Cu column. The results of the sequential Edman degradation of the 3H-labeled peptide are shown in Fig. 7. The amino acid sequence can be aligned with the predicted V-8 protease peptide extending from Val-93 to Glu-131 of the B subunit. Radiactivity was released at cycles 11 and 18, which correspond to Lys-103 and Lys-110. These amino acids were labeled to comparable extents, when corrected for the repetitive yield of 93%.

The possibility that the third lysine in this peptide, Lys- 129, was also labeled could not be excluded in this experiment, due to the considerable degeneration of the Edman degradation chemistry by the 37th cycle. The following experiment was designed to resolve this question. The labeled peptide spanning residues 93-131 was purified as above and digested with chymotrypsin. The digest was resolved on a Cq column into two peaks, designated C-l and C-2 (Fig. 8). The fractions from each peak were pooled separately and subjected to a final purification step on a reversed-phase Cu column. The amino acid sequence of peptide C-1 showed that it contained


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

21346 ATP Binding Site on DNA G-y-use

TABLE I Distribution of bound PLP-AMP between the gyrase A and B subunits

The gyrase-DNA complexes at 1 PM were reacted with the indicated concentrations of PLP-AMP, treated with 5 mM 13H]NaBH, (600 mCi/mmol), and freed of unbound radioactivity on spin columns. Aliquots were withdrawn for measurement of the stoichiometry of PLP-AMP binding. In sample 4, gyrase was incubated with ADP and DNA for 30 min prior to the addition of PLP-AMP. The samples (14 pg) were electrophoresedon a 7.5% acrylamide gel in the presence of SDS and the Coomassie Blue-stained bands were sliced for radioactive scintillation counting (see “Experimental Procedures”). The moles of incorporated analog associated with each subunit were based on the percentage of radioactivity in the subunits multiplied by the moles of incorporated PLP-AMP per mol of gyrase before electrophoresis. The stoichiometry data are expressed as moles of PLP-AMP/mol of gyrase A2BZ tetramer, or as moles of PLP-AMP/mol of A2 or B* dimer. The A and B subunits from the control enzyme that was not incubated with PLP-AMP retained minor amounts of radioactivity from the [3H]NaBH., treatment, in the amounts of 263 and 249 cpm, respectively. These background values were subtracted from the radioactivity associated with the corresponding subunits from gyrase samples incubated with PLP-AMP. - -

Sample

1 2 3 4

Additions PLP-AMP/gyI.WZ Gyrase A Gyrase B

PLP-AMP/AZ PLP-AMP/B, mol~nwl CPm % mol/mol CPm 70 mol/mol

3.2 PM PLP-AMP 0.43 354 9.5 0.04 1122 90.5 0.39 7.9 /.LM PLP-AMP 0.88 517 10.6 0.09 2392 89.4 0.79

18.4 PM PLP-AMP 1.67 822 13.7 0.23 3771 86.3 1.44 18.4 NM PLP-AMP + 2 mM ADP 0.65 713 28.5 0.19 1378 71.5 0.46

Fraction Number

FIG. 6. HPLC profile of V-8 peptides of gyrase modified by PLP-AMP. Gyrase was modified by [3H]PLP-AMP, digested with V-8 protease, and resolved on a Ca reversed phase column as described under “Experimental Procedures.” The eluate was monitored at 220 nm and by the radioactivity in 50-~1 aliquots from each 0.5-ml fraction.

residues 99-109, consistent with proteolytic cleavage after Leu-98 and Tyr-109. (The failure of chymotrypsin to cleave after Phe-104 can be explained by a marked decrease in the rate of bond hydrolysis caused by the neighboring acidic residues, Asp-105 and Asp-106 (Allen, 1981). In agreement with the results from Fig. 7, the radioactivity from peptide C- l was released at cycle 5 of the Edman degradation, which corresponds to Lys-103. For peptide C-2, the radiactivity was released at the first cycle. The yields of the phenylthiohydantoin-amino acids released during the sequencing run on peptide C-2 were unusually low, possibly due to the partial blockage of the Edman degradation caused by the pyridoxy- lated lysyl residue at the N terminus. Nevertheless, we were able to determine the sequence starting from the second cycle beginning with Val-111 and ending with Val-123, confirming Lys-110 as the labeled residue. It can be estimated, based on the repetitive yield of 93% during the sequence analysis of

0 I 0 5 10 15 20 25 30 35 40

Cycle Number

FIG. 7. Edman degradation of the 3H-labeled peptide generated from V-8 Droteolysis. The fractions containing the 3H- labeled peptide (centered at-fraction 117) in Fig. 6 were pooled and further purified on a Cls reversed-phase column before being sequenced (see “Experimental Procedures”). The letters at the top correspond to the amino acids identified as their phenylthiohydantoin derivatives. The dashes represent amino acids that were not detected by the analyzer due to low recovery near the end of the sequence analysis. Based on the alignment of this peptide with the known sequence of the B protein, the three dashes should correspond to Ser- 127, Leu-130, and Glu-131. The peptide is predicted to end at Glu- 131 from the known specificity of V-8 protease (Drapeau, 1977). The ordinate shows only a fraction of the total radioactivity released at each cycle of the Edman degradation since the majority of the solution was analyzed for amino acid identity. The repetitive yield during the sequence determination was 93%. The values on the ordinate were not corrected for this loss.

Fraction Number

FIG. 8. HPLC profile of a chymotryptic digest of the ‘H- labeled peptide, gyrase B peptide (93-131). The radiolabeled peptide was prepared as described in the legend to Fig. 6, digested with chymotrypsin, and resolved on a C, reversed-phase column (see “Experimental Procedures”). The radioactivity was determined in 15- ~1 aliquots from each l-ml fraction.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

ATP Binding Site on DNA Gyrase

gyrase B peptide (93-131), that the ratio of radioactivity associated with Lys-103 to Lys-110 was about 1.3; this value is close to the ratio of radioactivity in peptide C-l to peptide C-2 of 1.2. Thus, the similar ratios of radioactivity, together with the absence of a third peak containing radioactivity during the elution of the chymotryptic digest, suggest that Lys-129 was not significantly labeled.

DISCUSSION

We have used the nucleotide affinity analog PLP-AMP to label the ATP binding site on Escherichia coli DNA gyrase. Borohydride reduction of the Schiff base complex, formed between the 4-formyl group of the pyridoxyl moiety and a protein primary amino group, gave a stable derivative and allowed us to identify Lys-103 and Lys-110 as the sites of specific labeling on the B subunit. It is likely that the modified lysyl residues are at the ATP binding site from the following observations: 1) PLP-AMP is a potent inhibitor of both the ATPase and DNA supercoiling activities, with 50% inhibition observed at about 7.5 PM inhibitor. This concentration is about 40-fold lower than the K,,, for ATP, showing that PLP- AMP binds with considerable affinity. 2) The relationship between the stoichiometry of PLP-AMP binding and loss of ATPase activity is consistent with inactivation resulting from PLP-AMP binding to only one of two ATP binding sites. Because gyrase contains a total of 190 lysyl residues, the result shows that modification by PLP-AMP is highly selec- tive. 3) The inhibition of ATPase activity appears to be of the ail-or-none type because the K,,, for ATP of gyrase samples inhibited from 30 to 70% was the same as found for the unmodified control. This behavior is indicative of an inactivation process and is consistent with the modification at the ATP binding site. 4) Approximately 90% of the bound analog was found to be associated with the B subunit which carries the ATP binding site. The protection against modification caused by ADP binding was due to a diminished incorporation into the B subunit while the residual amount of analog associated with the A subunit was largely unchanged. 5) Lastly, the modified residues, Lys-103 and Lys-110, are in a region of the primary sequence that is well conserved among other type II topoisomerases (see below).

The reaction between PLP-AMP and gyrase is likely to involve an initial binding step, followed by formation of a reversible Schiff base complex with the t-amino group of a lysine. Treatment with NaBH, will stabilize the Schiff base complex but not the fraction bound as a noncovalent complex; this can explain the residual ATPase activity seen at nearly saturating concentrations of PLP-AMP (Fig. 1). Following removal of unbound inhibitor, the residual ATPase activity that appears resistant to PLP-AMP modification is likely to be dependent on the equilibrium constant for the Schiff base formation. This behavior has been observed in modification studies on several nucleotide binding enzymes employing pyridoxal 5’-phosphate (Chen and Engel, 1975), PLP-AMP (Tamura et aZ., 1986; 1988; Tagaya et al., 1988a; Miziorko et al., 1990), and related analogs (Tagaya et al., 1985; Tagaya and Fukui, 1986; Ohmi et al., 1988).

The nonlinear relationship between loss of ATPase activity and stoichiometry of PLP-AMP binding to gyrase (Fig. 3) suggests a complex mode of inhibition. Attempts were made to minimize enzyme heterogeneity in the reaction mixture by forming gyrase complexes with linear pUC9 DNA at concentrations giving maximal DNA-stimulated ATPase activity. Under these conditions, it is likely that all gyrase molecules are saturated with a single topological form of DNA. However, in view of the moderate but significant sequence specificity

for DNA cleavage (Morrison and Cozzarelli, 1979; Lockshon and Morris, 1985), it is improbable that all gyrase molecules are bound to strictly equivalent sites; this may account for some differences in reactivity. It can also be argued that the B subunits may not all be functionally equivalent during normal catalysis, because it is routinely found in gyrase A and B subunit preparations that the specific supercoiling activity of the B subunit is less than that of the A subunit (Mizuuchi et al., 1984).

Alternatively, the ATP hydrolysis activity of gyrase may require that both ATP binding sites are functional. Indeed, the experimental data fit closely to a computer-generated theoretical curve that assumes complete inactivation of the tetramer resulting from PLP-AMP binding to only one of two catalytic sites. This phenomenon is often observed with enzymes containing multiple copies of catalytic sites (see Lev- itzki and Koshland, 1976). For example, it was shown in a recent report (Dombroski et al., 1988) that complete inactivation of E. coli transcription termination factor rho results when about 3 molecules of PLP-AMP are bound to each enzyme hexamer.

It has been previously reported (Brown et al., 1979; Gellert et al., 1979) that a C-terminal 50,000 molecular weight proteolytic fragment of the B subunit could combine with the A subunit to give a DNA relaxing activity. Because this complex lacked all ATP-dependent functions, it seemed reasonable that at least part of the ATP binding site was contained in the lost N-terminal 392 amino acids (Adachi et al., 1987). Our current finding that Lys-103 and Lys-110 are labeled by PLP- AMP provides us with a more precise map of the N-terminal region that may function as part of the ATP binding domain. If we assume that PLP-AMP binds to gyrase in an extended conformation, the reactive 4-formyl group on the pyridoxyl moiety would occupy a position normally accommodating the y-phosphate of ATP. This suggests that the t-amino groups of Lys-103 and Lys-110 may be proximal to the phosphoryl groups of the bound ATP molecule. The results of the Edman degradation indicate that PLP-AMP is probably not bound to both Lys-103 and Lys-110 on the same peptide chain but rather on separate monomers. This conclusion is based on the observation that both radioactivity and the phenylthiohydantoin derivative of lysine are released simultaneously at the cycles corresponding to both positions 103 and 110. Although the close proximity of Lys-103 and Lys-110 on the primary sequence does not necessarily imply that they are spatially close, one can surmise that if both residues are at the active site, their labeling by PLP-AMP would be mutually exclusive.

The amino acid sequences of eight type II topoisomerases are known. The enzymes from eukaryotes contain sequences related to both the bacterial gyrase A and B subunits (Uemura et al., 1986; Lynn et al., 1986) as well as the T4 gene 39 and 52 proteins (Huang, 1986a, 1986b). The N-terminal part of the eukaryotic enzymes is related to the gyrase B subunit and the phage T4 gene 39 protein, while the central to C-terminal portion is similar to the gyrase A subunit and the phage T4 gene 52 protein. Fig. 9 shows an amino acid sequence alignment of residues 93-131 from the E. coli B subunit with seven other type II topoisomerases. Lys-110 is conserved in six of the eight enzymes; the remaining two proteins, the enzyme from Trypanosoma brucei and the T4 gene 39 product, contain asparagine and a conservative replacement, arginine, respectively. On the other hand, Lys-103 is conserved only in the B subunit from Bacillus subtilis, with asparagine in this position in the other six cases. A closer inspection of the sequence alignments reveals five conserved amino acids on the C- terminal side of the PLP-AMP-modified lysines, of which


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

21348 ATP Binding Site on DNA Gyrase

FIG. 9. Amino acid sequence alignment of the PLP-AMP labeled V-8 peptide with homologous regions from other type II topoisomerases. The sequences are labeled as follows: &o-B, Escherichia coli gyrase B protein; Bsu-B, Bacillus subtilis gyrase B protein; T4-39, phage T4 gene 39 protein; See, Saccharomyces cere- visiae; Spo, Schizosaccharomyces pombe; Tbr, Tryparwsoma brucei; Dme, Drosophila mekmogaster; Hum, human. The alignments of Eco- B, Bsu-B, T4-39, See, Spo, and Dme were taken directly from Wyckoff et al. (1989). (A misprint at position 111 on the &o-B sequence has been corrected from methionine to valine to agree with the original sequence reported by Adachi et al., 1987). The sequences of Tbr (Strauss and Wang, 1990) and Hum (Tsai-Pflugfelder et al., 1988) were aligned by visual inspection. The amino acid residues in bold letters indicate positions where all eight enzymes are identical. The boxes emphasize homologous regions where 6 or more residues are identical or contain conservative substitutions. The asterisks above lysines 103 and 110 of Eco-B mark the residues labeled by PLP-AMP.

three are glycine at positions 114,117, and 119. An interesting possibility is that this region may serve an analogous function to the “glycine-rich flexible loop” (consensus sequence A from Walker et al., 1982 as modified by Chin et al., 1988) found in many, but not all, ATP binding enzymes. Some ATP binding enzymes have been reported to have A-like consensus sequences while others, particularly some of the glycolytic kinases, show no obvious homology with this region (Serrano, 1988). In several well characterized enzymes, this glycine-rich region has been proposed to undergo conformational changes that lead to interactions of catalytic residues with the phosphoryl groups of bound ATP (Mildvan and Fry, 1987; Serrano, 1988). Additional support for the importance of this region has emerged from studies demonstrating that lysyl residues within this flexible loop structure have been targets for modification by PLP-AMP in rabbit muscle adenylate kinase (Tagaya et al., 1987) and E. coli transcription termination factor rho (Dombroski et al., 1988). Furthermore, the ras oncogene product p21 (Ohmi et al., 1988) and the E. coli F1- ATPase /3 subunit (Tagaya et al., 1988b) were both specifically labeled by related analogs at the lysyl residue homologous to the one modified by PLP-AMP in adenylate kinase. Prior to our chemical modification data, it was suggested that residues 125 to 142 of the T4 gene 39 protein may be part of an ATP binding fold based on their limited similarity to consensus sequence A (Huang, 1986a). The V-8 peptide Gyr B(93-131), containing the two modified lysyl residues, overlaps with this region of the gene 39 protein and supports this earlier predic- tion.

A second less restrictive consensus pattern, (R/K/H) (X,,)~X@#J(E/D), where 4 are hydrophobic amino acids, also occurs in many ATP binding enzymes downstream from consensus sequence A (Chin et al., 1988). The acidic residue at the end of this segment is believed to function in binding the phosphoryl groups of ATP through a chelated magnesium in adenylate kinase (Mildvan and Fry, 1987), and in the related sequence patterns in phosphofructokinase and phos- phoglycerate kinase (see Serrano, 1988). We have identified a sequence similar to the above motif in the eight type II topoisomerases. In the E. coli B subunit, it spans residues 413-424. The sequence pattern is as follows: (R/K)(X&L( Y/ F/V/I)(L/I/V)(V/T)IJ where the conserved amino acids are underlined. The four amino acids following the glutamic acid residue at position 424, GDSA, are invariant among the type

II topoisomerases, indicating that this segment constitutes another highly conserved region. Substitution of asparagine for aspartic acid at position 426 (nul-24 mutation) confers - resistance to the antibiotic, nalidixic acid (Yamagishi et al., 1986). The importance of this region in ATP binding remains to be experimentally determined, however.

To date, several functional domains in gyrase have been identified. Limited tryptic digestion of the A subunit generated a 64-kDa fragment from the N terminus which still retained its ability to combine with the B protein and carry out all known activities of the native enzyme (Reece and Maxwell, 1989). Although the absence of the C-terminal 33- kDa region of the A subunit resulted in a diminished stability of the enzyme-DNA complex, it was clear that a functional site involved in DNA cleavage was still present. This is consistent with an earlier finding that Tyr-122 of the A subunit forms a covalent 5’-phosphoryl linkage with the DNA (Horowitz and Wang, 1987). Thus, the N-terminal region of the A protein must contain sites for interaction with the B subunit to form an active enzyme complex. Likewise, the fragment of the B subunit which lacked the N-terminal 392 amino acids still retained contact sites for combining with and activating the A subunit (Gellert et al., 1979; Brown et al., 1979; Adachi et al., 1987). From our results, we can now more precisely assign a region within the N terminus of the B subunit that may function as part of the ATP binding domain. Site-directed mutagenesis studies may provide clues to possible functional roles for Lys-103 and Lys-110.

Acknowbdgments-We are grateful to Dr. Andrew D. Bates, Dr. Judah L. Rosner, and Mary H. O’Dea for helpful discussions and comments on the manuscript.

REFERENCES

Adachi, T., Mizuuchi, M., Robinson, E. A., Appella, E., O’Dea, M. H.. Gellert. M., and Mizuuchi, K. (1987) Nucleic Acids Res. 16, 771-784

Allen, G. (1981) in Sequencing of Proteins and Peptides (Work, T. S., and Burdon. R. H.. eds) DD. 43-71. Elsevier/North-Holland Biomedical Press, Amsterdam -

Brown, P. O., Peebles, C. L., and Cozzarelli, N. R. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 6110-6114

Chen, S.-S., and Engel, P. C. (1975) Biochem. J. 149,627-635 Chin, D. T., Goff, S. A., Webster, T., Smith, T., and Goldberg, A. L.

(1988) J. Biol. Chem. 263,11718-11728 Dombroski, A., LaDine, J. R., Cross, R. L., and Platt, T. (1988) J.

Biol. Chem. 263,18810-18815 Drapeau, G. R. (1977) Methods Enzymol. 47,189-191 Fisher, L. M., Mizuuchi, K., O’Dea, M. H., Ohmori, H., and Gellert,

M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,4165-4169 Gellert, M., Fisher, L. M., and O’Dea, M. H. (1979) Proc. Natl. Acad

Sci. U. S. A. 76,6289-6293 Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem.

267,12092-12100 Horowitz, D. S., and Wang, J. C. (1987) J. Biol. Chem. 262, 5339-

5344 Huang, W. M. (1986a) Nucleic Acids Res. 14,7751-7765 Huane. W. M. (1986133 Nucleic Acids Res. 14,7379-7390 Kirkegaard, K.;and Wang, J. C. (1981) Cell 23, 721-729 Klevan, L., and Wang, J. C. (1980) Biochemistry 19,5229-5234 Laemmli, U. K. (1970) Nature 227,680-685 Levitzki. A.. and Koshland, D. E. (1976) Curr. Top. Cell. Regul. 10,

l-40 ' Liu, L. F., and Wang, J. C. (1978a) Cell 15, 979-984 Liu, L. F., and Wang, J. C. (197813) Proc. Natl. Acad Sci. U. S. A.

75,2098-2102 Lockshon, D., and Morris, D. R. (1985) J. Mol. Biol. 181, 63-74 Lynn, R., Giaever, G., Swanberg, S. L., and Wang, J. C. (1986)

Science 233,647-649 Maxwell, A., and Gellert, M. (1986) Adv. Protein Chem. 38, 69-107 Maxwell, A., and Gellert, M. (1984) J. Biol. Chem. 259,14472-14480


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

ATP Binding Site on DNA Gyrase 21349

Mildvan, A. S., and Fry, D. C. (1987) Adv. Enzymol. Relat. Areas. Mol. Biol. 58,241-313

Miziorko, H. M., Brodt, C. A., and Krieger, T. J. (1990) J. Biol. Chem. 265,3642-3647

Mizuuchi, K., O’Dea, M. H., and Gellert, M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75,5960-5963

Mizuuchi, K., Mizuuchi, M., O’Dea, M. H., and Gellert, M. (1984) J. Biol. Chem. 259,9199-9201

Morrison, A., and Cozzarelli, N. R. (1979) Cell 17, 175-164 Morrison, A., and Cozzarelli, N. R. (1981) Proc. N&l. Acad. Sci. U.

S. A. 78,1416-1420 Ohmi, N., Hoshino, M., Tagaya, M., Fukui, T., Kawakita, M., and

Hattori, S. (1988) J. Biol. Chem. 263, 14261-14266 Penefsky, H. S. (1977) J. Biol. Chem. 252,2891-2899 Rao. R.. Cunnineham. D.. Cross. R. L.. and Senior. A. E. (1988) J.

Bk. &em. 283,5640-5645 ’ ’ Rau, D. C., Gellert, M., Thoma, F., and Maxwell, A. (1987) J. Mol.

Biol. 193,555-569 Reece, R. J., and Maxwell, A. (1989) J. Biol. Chem. 264, 19648-

19653 Serrano, R. (1988) Biochim. Biophys. Acta 947, l-28 Staudenbauer, W. L., and Orr, E. (1981) Nucleic Acids Res. 9, 3589-

3603 Strauss, P. R., and Wang, J. C. (1990) Mol. Bioch.em. Parasitol. 38,

141-150 Sugino, A., Higgins, N. P., Brown, P. O., Peebles, C. L., and Cozzarelli,

N. R. (1978) Proc. Natl. Acad. Sci. U. S. A. 75.4838-4842 Sugino, A., and Cozzarelli, N. R. (1980) J. Biol.. Chem. 255, 6299-

6306

Swanberg, S. L., and Wang, J. C. (1987) J. Mol. Biol. 197,729-736 Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964 Tagaya, M., Hayakawa, Y., and Fukui, T. (1988a) J. Biol. Chem. 263,

10219-10223 Tagaya, M., Noumi, T., Nakano, K., Futai, M., and Fukui, T. (1988b)

FEBS Z&t. 233,347-351 Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260,

6670-6676 Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262,

8257-8261 Tamura. J. K.. Rakov. R. D.. and Cross. R. L. (1986) J. Biol. Chem.

261,4126-4133 ’ Tamura, J. K., LaDine, J. R., and Cross, R. L. (1988) J. Biol. Chem.

263.7907-7912 Tanford, C., and Roberts, G. L., Jr. (1952) J. Am. Chem. Sot. 74,

2509-2515 Tsai-Pflugfelder, M., Liu, L. F., Liu, A. A., Tewey, K. M., Whang-

Peng, J., Knutsen, T., Huebner, K., Croce, C. M., and Wang, J. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,7177-7181

Uemura, T., Morikawa, K., and Yanagida, M. (1986) EMBO J. 5, 2355-2361

Vosberg, H.-P. (1985) Curr. Top. Microbial. Zmmunol. 114,19-102 Walker, J. E., Sara&e, M., Runswick, M. J., and Gay, N. J. (1982)

EMBO J. 1,945-951 Wang, J. C. (1985) Annu. Rev. Biochem. 64,665-697 Wang, J. C. (1987) Biochim. Biophys. Acta 909, l-9 Wyckoff, E., Natalie, D., Nolan, J. M., Lee, M., and Hsieh, T.-S.

(1989) J. Mol. Biol. 205, 1-13 Yamagishi, J.-I., Yoshida, H., Yamayoshi, M., and Nakamura, S.

(1986) Mol. Gen. Genet. 204,367-373


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

J K Tamura and M Gellert5'-diphospho-5'-adenosine.

labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity

1990, 265:21342-21349.J. Biol. Chem.

http://www.jbc.org/content/265/34/21342Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/265/34/21342.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/265/34/21342

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;265/34/21342&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/265/34/21342

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=265/34/21342&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/265/34/21342

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/265/34/21342.full.html#ref-list-1

http://www.jbc.org/

characterization of the atp binding site on escherichia coli dna

Documents