Polyphosphate Kinase: Distribution, Someproperties and Its Application

Agric. Biol. Chem., 52 (6), 1471 - 1477, 1988 1471 Polyphosphate Kinase: Distribution, Some Properties and Its Application as an ATPRegeneration System Kousaku Murata,* Tomofumi Uchida, Jyoji Kato and Ichiro Chibata Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532, Japan Received December 23, 1987 Thedistribution and someproperties ofpolyphosphate kinase, that catalyzes the formation of polyphosphate from ATP, were investigated. High enzymeactivity was found in Alcaligenes faecalis, Brevibacterium ammoniagenes, Escherichia coli, Micrococuss lysodeikticus and Pseudomonasaeruginosa. The enzymerequired Mg2+for maximumactivity and was activated by basic proteins, polyamines and phosphate polymers of low molecular weight. The enzymefrom E. coli B could catalyze the reverse reaction, and generated ATPfrom ADPand metaphosphate. The feasibility of the generation of ATPby the E. coli B enzyme was confirmed by means of the coupled reactions of polyphosphate kinase and hexokinase, which produces glucose-6-phosphate. Wehave been studying phosphate polymer- polyphosphate kinase, that catalyzes the for- utilizing enzymes in microbial cells and have mation of highly polymerized phosphates from found that such enzymesare frequently found the terminal phosphate of adenosine-5'- in cells of Achromobacter, Brevibacterium and triphosphate (ATP) (Eq. I). This enzyme ac- Micrococcus species.1>2) Polyphosphate gluco- tivity has been found in various microbial kinase in Achromobacter butyri was used for strains such as Escherichia coli,6) Corynebac- the production of glucose-6-phosphate (G-6- terium xerosis1] and Enterobacter aerogenes.8) P) from glucose and metaphosphate.3) Although the reaction (Eq. I) is probably a Metaphosphate-dependent nicotinamide adenine dinucleotide (NAD) kinase in Brevi- ATP+ [Pi]n >ADP+ [PiL+1 (I) bacterium ammoniagenes4)was also useful for biosynthetic route, the enzyme in E. coli has the production of nicotinamide adenine dinu- been shown to catalyze the phosphorylation of cleotide phosphate (NADP)from NADand adenosine-S'-diphosphate (ADP) using phos- metaphosphate.5) The activities of poly- phate polymers6) (Eq.II). The results suggested phosphate fructokinase, polyphosphate man- that nokinase and enzymes catalyzing the phosphorylation of purine and pyrimidine nucleosides ADP+ [Pi]n >ATP+ [Pi]II_1 (II) to the corresponding nucleotides using meta- the polyphosphate kinase may be applicable to phosphate as a phosphoryl donor were also a bioreactor system as an ATPregeneration detected in cell extracts prepared from system, since substrates (phosphate polymers) Enterobacter aerogenes, Brevibacterium ammo- for the enzyme are readily available at low niagenes, Micrococcus lysodeikticus and other cost. strains (Murata et al, unpublished data). To determine the feasibility of the polyphos- Other than these phosphate polymer- phate kinase reaction as an ATPregeneration utilizing enzymes, microorganisms contain system, we investigated the distribution and Present address: Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan. 1472 K. Murata et al. some properties of the enzyme in micro- fraction." The SI fraction contained 25mMacid labile organisms. phosphate, which was estimated by determining the inorganic phosphate concentration after hydrolysis of the phosphate polymers in 1.0n HC1 at 100°C for 7min. MATERIALS AND METHODS Inorganic phosphate was determined by the method of Fiske and Subbarow.10) Preparation of cell extracts. All microbial cells were grown in a medium containing 0.5% glucose, 1.0% yeast Assay for polyphosphate kinase. The activity as to the extract, 1.0% polypeptone, 0.2% meat extract, 0.5% forward reaction (Eq. I) of this enzyme was assayed in a KH2PO4 and 0.5% NaCl (pH 7.2). The cultures were reaction mixture (0.1 ml) containing 0.2fimo\ MgCl2, reciprocally shaken at 30°C for 16hr in 100ml medium in 100 fig protamine, 0.04^mol y-32P-ATP (0.4 fid), 5.0 fiinol a 2 1 Sakaguchi flask. The harvested cells were washed potassium phosphate buffer (pH 7.2) and cell extract once with a 0.85%saline solution and then resuspended in (10fig as protein). The reaction was carried out at 30°C for 5.0raM potassium phosphate buffer (pH 7.2) containing 10min and then aliquots (20/il) of the reaction mixture 0.5mM sodium deoxycholate. The cell suspension was were spotted onto 2cm squares ofToyo Filter Paper (No. ultrasonically treated at 90kHz for 5min in a Kubota 51). Chromatography was conducted at room temperature Model 200M Insonator and then the supernatant obtained by the descending method with 10% trichloroacetic acid on centrifugation at 25,000xg for 40min was dialyzed (TCA) as a solvent. After development, the paper was against 5.0mMpotassium phosphate buffer (pH 7.2) at dried and then the origins were cut out in 2cm squares for 4°C overnight. Protein was determined by the method of radioactivity determination. The activity as to the reverse Lowry et al.9) reaction (Eq. II) of this enzyme was determined in a reaction mixture (0.1 ml) containing 2.0/imol (as acid Partial purification ofpolyphosphate kinase from E. coli labile phosphate) SI fraction, 0.5/imol MgCl2, 0.2/imol B. To a cell suspension (25ml, 0.5g cells/ml) ofE. coli B, ADP, 5.0fimo\ potassium phosphate buffer (pH 7.2) and 2.0mg lysozyme was added. After incubation at 37°C for the partially purified polyphosphate kinase (10 fig as pro- 30min, 0.1mg DNase and 0.05mg RNase were added tein) from E. coli B. After incubation at 30°C for several successively to the mixture and then the incubation was hours, the reaction was terminated by immersing the test continued for a further 30min. To the mixture was then tube in boiling water for 1min, and then ATP in the added 1.0mg streptomycin sulfate, and the precipitated supernatant was determined by means of the Luciferase- material was collected by centrifugation at 25,000 x g for Luciferin assay. U) 30 min. The precipitate was dissolved in 5.0 mMpotassium phosphate buffer (pH 7.2) (25ml, 2.1 mg/ml protein) and Coupled reactions of polyphosphate kinase and hexo- then fractionated with ammonium sulfate. Solid am- kinase. To produce G-6-P from glucose, the ATP gener- moniumsulfate (7 g) was added to the streptomycin sulfate ation reaction (reverse reaction) catalyzed by polyphos- fraction, followed by incubation at 0°C for 30min. The phate kinase was coupled with the hexokinase reaction. precipitate was obtained by centrifugation at 25,000 x g The reaction mixture (0. 1 ml), consisting of2.0 /imol ADP, for 30min, dissolved in 2.5ml of 5.0mM potassium phos- 5.0/imol glucose, 2.0/imol (as acid labile phosphate) SI phate buffer (pH 7.2) and then dialyzed against the same fraction, 0.5 /imol MgCl2, 5.0 /imol potassium phosphate buffer at 4°C overnight. The dialysate thus obtained buffer (pH 7.2), 5.0 units of hexokinase and 10fig (as (3.2ml, 2.4mg protein) was used as the source of poly- protein) of the partially purified polyphosphate kinase phosphate kinase throughout this study. Through the from E. coli B, was incubated at 30°C for several hours. purification steps described above, an approximately 15- The reaction was terminated by boiling at 100°C for 1 min fold increase in the specific activity of polyphosphate and then G-6-P in the supernatant was determined en- kinase wasattained. zymatically by the method of Hohorst.12) Columnchromatography of metaphosphate. The com- Chemicals. Polyphosphate and metaphosphate were ponents in metaphosphate were fractionated by Dowex purchased from Katayama Chemicals Industry, Co., Ltd., 1 x2 (Cl~) column chromatography as described pre- Osaka, Japan. Ribonuclease, deoxiribonuclease, trimeta- viously.l) The metaphosphate solution (10%, pH 6.5) was phosphate and tetrametaphosphate were from Sigma applied to the column (5cm x 30cm) and then the column Chemical Co., St. Louis, MO. y-32P-Adenosine 5'-tri- was extensively washed with water. The absorbed phos- phosphate was purchased from New England Nuclear, phate polymers were then eluted with a linear gradient of Mass. LiCl, the concentration increasing from 0 (400ml) to 1.0 m (400ml) (pH 7.2). Fractions were collected at 7ml/tube/ RESULTS 5min at room temperature. The active fractions (Nos. 48 ~52), as a phosphoryl donor for polyphosphate kinase Assay conditions for polyphosphate kinase in E. coli B, were pooled (35ml) and designated the "SI The assay conditions for polyphosphate ki- ATPRegeneration by Polyphosphate Kinase Reaction 1473 Fig. 1. Chromatographic Separation of 32P-ATP from 32P-polyphosphate. A: A chromatogram of a 20/il aliquot of the polyphosphate kinase reaction mixture incubated at 30°C for lOmin. B: Achromatogram of the corresponding control Fig. 2. Chromatographic Analysis of the Reaction using the boiled enzyme solution. C: A chromatogram of Products. the reaction mixture incubated in the absence of Mg2+. The polyphosphate kinase reaction (forward) was carried After development, the chromatographic paper was dried out as described under Materials and Methods. A: An and then cut into 1 cm sections except for the origins which aliquot (10/il) of the reaction mixture was spotted on to werecut out in 2cm squares for radioactivity determi- Toyo Filter Paper (No. 51) and then developed with Ebel's nation. Other conditions for the reaction and chromatog- solvent at room temperature. Ebel's solvent contained raphy are given under Materials and Methods. 350ml iso-propanol, 20g TCA, 2.5ml 25% NH4OHand 150ml of water. The chromatogram (ascending) was nase activity (Eq. I: forward reaction) were developed for approximately 20 hr. To locate the positions of authentic phosphate compounds, the chromatographic invetsigated using cell extract of Arthrobacter papers were sprayed with acid molybdate solution, heated atrocyaneus ATCC13752 showing poly- at 80°C for lOmin and then exposed to ultraviolet ra- phosphate kinase activity. Figure 1 shows diation. For the determination of radioactivity, the chro- the chromatographic patterns of the reaction matographic papers were dried and cut into 0.5cm sections, and then the radioactivity in each section was products.

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