Biochem. J. (1971) 125, 531-539 531 Printed in Great Britain Pyruvate,Phosphate Dikinase from Bacteroides symbiosus By RICHARD E. REEVES Department of Biochemi8try, Loui8iana State Univer&Uy School of Medicine, New Orleans, La. 70112, U.S.A. (Received 9 July 1971) 1. An improved method is given for preparation of pyruvate,phosphate dikinase from Bacteroidec 8ymbio8sq. 2. The bacterial enzyme is stable, free from interfering enzyme activities, and does not require thiol compounds to maintain stability during storage or assay. 3. New direct assays of enzyme activity are based on acid evolution or consumption as measured at constant pH in apH-stat. 4. The optimum rate of reaction in the direction of pyruvate formation occurs at about pH 6.4; in the direction of phosphoenolpyruvate formation, it is at pH 7.2-7.8. 5. Newly determined substrate Km values for the enzyme are: AMP, 3.5 x 10-6M; ATP, 1 X 10-4M; pyruvate, 8 x 10-5M; Pi, 6 x 10-4M. 6. K+ may substitute for NH4+ in activating the reaction catalysed by the B. symbio&wu enzyme. 7. In the direction ofpyruvate formation the bivalent metal ion requirement ofthe enzyme is fulfilled by salts of nickel, manganese, magnesium and cobalt. In the other direction only magnesium salts were effective. 8. The nucleotide specificity of the enzyme is strictly limited to the adenine nucleotides. CTP and ITP strongly inhibit the reaction in the direction of phosphoenolpyruvate formation. The enzyme now called by the trivial name The present paper reports further work on the pyruvate,phosphate dikinase was first reported by preparation and properties of the enzyme from B. Hatch & Slack (1968) in plant sources. Reeves 8ymbio8u8. Enzyme from this source is very stable, (1968) found the enzyme in Entamoeba hi8tolytica. does not require thiols, and is readily prepared in Reeves, Menzies & Hsu (1968), Evans & Wood good yield in a state free from interfering enzyme (1968) and Benziman & Palgi (1970) identified it in activities. Additionally, methods of asay are bacterial sources. Reeves et al. (1968), working with introduced that are based on H+ consumption or the enzyme from Bacteroidec 8ymbio8u8, established evolution, according to the above equation. These that label from [32P]P? appears in the y-position methods avoid the restrictions imposed by linked- of ATP and labelled phosphate from phospho- enzyme systems and they offer advantages over enol pyruvate appears in the ,-position. They also fixed-term assays of enzyme products. found the observed equilibrium constant in the direction of pyruvate formation to be 1140 at pH 7 and that, over a wide pH range, it varied directly MATERIALS with the square of the H+ concentration. This indicates that two hydrogen ions are consumed or Bacteroides 8ymbio8su (A.T.C.C. 14940) was grown produced in the forward or reverse reactions, anaerobically in 400-litre lots at the Fermentation Plant of the Department of Biochemistry, University of Georgia, respectively. The reaction that occurs the pres- Athens, Ga., U.S.A. The medium contained (g/l): ence of Mg2+ was written as follows: Tryptone (Difoo-0123) (20), D-gluoose (10), yeast extract 2H+ +AMP2- + MgPP12- + phosphoenolpyruvate3 (Difoo) (2), NaCl (2.5), anhydrous K2HPO4 (1.5), mer- MgATP2-+P12-+pyruvate- captosuccinic acid (1.5), L-cysteine hydrochloride (0.79) and NaOH to pH7.0. Incubation was for 16-20h at Andrews & Hatch (1969) and Evans & Wood 370C. Cells were harvested on a Sharples supercentrifuge, (1971) have undertaken studies on the mechanism frozen in solid C02, and stored at -60°C until used. The of action of dikinase by using isotope-exchange yield of wet cell paste was approx. 5.7g/l. reactions. These workers noted certain difficulties Nucleoside mono- and tri-phosphates and the pyridine nucleotides were obtained from P-L Laboratories, with enzyme prepared from plant sources and Milwaukee, Wis., U.S.A. Sigma Chemical Co., St Louis, propionibacteria. The enzyme was cold-labile and Mo., U.S.A., supplied hexokinase, phosphoenolpyruvate required the presence of thiol compounds during (tricyclohexylammoniumsalt)andDEAE-eellulose. Lyso- storage and assay. zyme was from Nutritional Bioehemioals Inc., Cleveland, 532 R. E. REEVES 1971 Ohio, U.S.A. Bio-Gel P-300 was from Bio-Rad Labora- gradient achieved by allowing buffer containing 0.5m- tories, Richmond, Calif., U.S.A. Mercaptosuccinic acid NaCl to flow into the closed reservoir containing 600ml of and pyruvic acid were from Eastman Chemical Co., 75 mm-NaCl-buffer. The effluent was collected in frac- Rochester, N.Y., U.S.A. The pyruvic acid was distilled tions of 36ml. Enzyme was eluted immediately after the before use. Glucose 6-phosphate dehydrogenase and highly coloured material. Fractions 19-23 comprising lactate dehydrogenase were from Boehringer und Sohne, most of the enzyme peak were combined and dialysed Mannheim, Germany. Inorganic pyrophosphatase was against buffer. prepared from E8cherichia coli. Crude enzyme was The dialysed solution from the first column was placed prepared by the method of Blumenthal, Johnson & on a second column prepared from 20g of DEAE-cellulose, Johnson (1967). It was concentrated by vacuum dialysis similarly preconditioned. The enzyme solution was against lOmx-tris-HCl buffer, pH7.2, containing 1mM- applied and the column washed with 300 ml of the 75 mm- MgSO4. It was purified by chromatography on a column NaCl-buffer. Enzyme was eluted with a linear gradient containing BOg of Sephadex G-25 (Pharmacia), by elution prepared by allowing 600ml of0.4M-NaCl in buffer to flow with the same buffer. The peak fractions of enzyme from into an open reservoir containing an equal volume of the column were used. The inorganic pyrophosphatase 75mm-NaCl-buffer. Effluent was collected in 32ml was free from non-specific phosphatases. fractions. Enzyme appeared in fraction 14, immediately DEAE-cellulose was prepared for column use by after a slightly coloured material. Three fractions com- allowing 20g to sink in 1 litre of 1 m-NaOH. Then 750g of prising the enzyme peak were combined, and enzyme was cracked ice and 250ml of conc. HCI were added, with precipitated by stirring in solid (NH4)2SO4 (45g/100ml). cooling and stirring. The suspension, diluted to 4 litres The suspension was centrifuged in the cold and most ofthe with cold water, was washed by decantation until the supernatant solution was withdrawn and discarded. The free acid concentration fell below 0.01 m. Concentrated sediment was suspended in the remaining supernatant solutions of imidazole-HCl buffer, pH7, NH4C1, NaCI solution (11.5ml) and stored in the refrigerator. Portions and EDTA (sodium salt) were then added to final con- of this suspension were centrifuged. The sediment was centrations of 20, 20, 75 and 1 mm, respectively, and the dissolved in buffer, and dialysed twice against 300 vol. of suspension was adjusted to pH7 with NaOH. buffer. Such enzyme is referred to as enzyme from the second (NH4)2SO4 precipitation. A 4.5 ml portion ofthe above suspension was centrifuged. METHODS The sediment was dissolved in buffer and dialysed against 100vol. of buffer. This solution was placed on a column Enzyme assays were done at 25BC. The enzyme puri- prepared from lOg of Bio-Gel P-300 that had previously fication procedures were done at 0-40C except where been conditioned with SOOml of buffer. The sample otherwise noted. The buffer contained 20mm-imidazole volume was 6ml. It was eluted with buffer and collected base adjusted to pH7 with HCI, 20mm-NH4Cl and in 6ml fractions. Enzyme appeared in fractions 17-25. 1 mm-EDTA (sodium salt). All modifications of this Enzyme activity and E280 were parallel across the enzyme buffer are specifically noted in the text. peak. The Bio-Gel column was prepared and used at Extraction and purification of B. symbiosus dikinase. room temperature. This enzyme is referred to below as Frozen cells (107g) were thawed and washed by centri- column-purified enzyme. fugation with 1.5 litres of 0.15M-NaCl. The packed cells Enzyme a88ay8. Assay A. The standard enzyme assay were resuspended in 2 litres of buffer containing 5mM- was slightly modified from that described by Reeves et al. EDTA (sodium salt). The suspension was incubated at (1968). Cuvettes contained 50mM-imidazole-HCl buffer, 370C while 100mg of separately dissolved crystalline pH6.8; 5mM-MgCl2; 2OmM-NH4Cl; 1mm-AMP; lmM- lysozyme was added with stirring. Cell lysis was apparent phosphoenolpyruvate; 0.2 mM-NADH2; 4 units of lactate within 15min. After 35min the viscous suspension was dehydrogenase; and about 0.01 unit ofenzyme in a volume chilled in an ice bath and a solution containing lOg of of 0.39 ml. After monitoring the change in extinction at streptomycin base, as the sulphate, was added dropwise 340 nm for a few moments reaction was started by the with stirring. The suspension was centrifuged for 10min addition of 0.01 ml of 50mM-sodium pyrophosphate. The at 9000g and the sediment was discarded. To the super- final rate of extinction change was corrected for any rate natant solution was added 450g ofsolid (NH42004/1, with noted before the addition ofthe PP1. Such corrections were stirring. The precipitate was collected by centrifugation not needed with enzyme preparations obtained after the and redissolved in 204ml of buffer. stage of the second DEAE-cellulose-column step. One To the above solution was added sufficient 4M- unit ofenzyme activity is defined as the amount that would potassium acetate to make the concentration 0.05m with effect the oxidation of 1,mol of NADH2/min under the respect to acetate. The solution was chilled in an ice bath conditions of the assay. The molar extinction of NADH2 while 2m-acetic acid was added dropwise with stirring was taken to be 6.22 x 1031-molh-Icm-2 at 340nm.