Phosphorylation Enzymes of the Propionic Acid Bacteria and The

Proc. Natl. Acad. Sci. USA Vol. 82, pp. 312-315, January 1985 Biochemistry Phosphorylation enzymes of the propionic acid bacteria and the roles of ATP, inorganic pyrophosphate, and polyphosphates (PP, phosphofructokinase/polyphosphate glucokinase/fermentation mechanism/polyphosphate kinase/phosphoglycerate kinase) HARLAND G. WOOD* AND NEIL H. GOSS*t *Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106; and tBiotechnology, 28 Barcoo Street, Roseville, Australia 2069 Contributed by Harland G. Wood, September 13, 1984 ABSTRACT It is shown that polyphosphates are not gen- This finding raises the possibility that P. freudenreichii phos- erated in significant amounts in the phosphoglycerate kinase phorylates glucose almost exclusively with poly(P). In addi- reaction; polyphosphate is more effective than ATP in the for- tion, Kulaev et al. (4) find that Propionibacterium shermanji mation of glucose 6-P by glucokinase, but the rate with ATP catalyzes Reaction 6. polyphosphate may be adequate to meet the requirements of glucose metabo- 3-phosphoglycerate lism; PP1 is far more effective than ATP as a phosphate donor kinase in the formation of fructose 1,6-P2 by phosphofructokinase; PPj rather than ATP almost certainly is used in this reaction; 1,3-diphosphoglycerate + poly(P)X, = and, aside from glucokinase and phosphofructokinase, the en- 3-phosphoglycerate + poly(P).+1 16] zymes of phosphorylation are specific in their requirements of phosphate donors or acceptors and are present in adequate amounts to meet the requirements of glucose metabolism by More recently, Bertagnolli and Cook (6) have reported that the propionic acid bacteria. poly(P)3 and poly(P)4 function in addition to PPj as phosphate donors in Reaction 3. These latter findings made us The propionic acid bacteria are unusual in that they utilize consider that poly(P) or PPj might be active in reactions of PP, in several phosphorylation reactions in place of ATP (1, the propionic acid fermentation not previously considered. 2). We, therefore, undertook a general survey of the enzymes to determine their carboxytrans- catalyzing phosphorylation reactions phosphorylase specificity for ATP, PPj, and poly(P). Most studies of the enzymes of the propionic acid bacteria have been done with oxalacetate + PPi = , P. shermanii and in fact, transcarboxylase, a central enzyme P-enolpyruvate + CO2 + Pi [1] of this fermentation, has not been demonstrated in other spe- cies. It was therefore considered of value to include this en- pyruvate, zyme in the study. For a recent review of the metabolism of phosphate dikinase the propionic acid bacteria, see ref. 7. P-enolpyruvate + PP1 + AMP ' pyruvate + P1 + ATP [2] MATERIALS AND METHODS Cultures and Conditions for Growth. Propionibacterium pyrophosphate phosphofructokinase arabinosum, ATCC 4965; P. freudenreichii, ATCC 6207; and P. shermanii, 52W were investigated. The latter was orig- fructose 6-P + PP1 ' inally isolated by Virtanen (8) and was later given the num- fructose 1,6-P2 + [3] ber 52W by C. H. Werkman (Iowa State University). The Pi bacteria were grown at 30°C in 15 liters of medium containing glucose, 45 g; Na2CO2, 82.5 g; KH2PO4, 153 g; yeast ex- Uryson and Kulaev (3) have shown that these bacteria also tract, 76 g; Co(NO3)266H2O, 0.15 g; thiamin HCl, 15 mg; utilize inorganic polyphosphate [poly(P)"] to phosphorylate calcium pantothenate, 15 mg; biotin, 7.5 mg; and 20 ml of glucose (Reaction 4) and Kulaev et al. (4) and Robinson et trace elements (boric acid, 75 mg; CuS04, 6 mg; Nal, 13.5 al. (5) have shown that they form poly(P) from ATP (Reac- mg, FeSO4, 66.2 mg; ZnCl2, 28.4 mg; and SeO2, 16.6 mg per tion 5). 100 ml). Glucose and Na2CO3 were each sterilized separate- polyphosphate ly. Glycerol medium was identical except 450 g of glycerol glucokinase was substituted for glucose. Lactate medium contained (per glucose + poly(P)n - 15 liters) sodium lactate (USP 60% syrup, Pfansted Labora- tories, Worthington, IL), 750 ml; yeast extract, 60 g; tryp- glucose 6-P + poly(P),,1 [4] tone, 30 g; K2HPO4, 60 g; NaH2PO4, 30 g; FeSO4, 0.15 g; polyphosphate and CoNO3, 0.15 g; vitamins and trace elements were as giv- kinase en above for the glucose medium. Bacteria were harvested ATP + poly(P),' , ADP + poly(P),+1 [5] using a Sharples centrifuge, either after 4-6 days during the stationary phase of growth (Table 1) or after 2, 4, and 7 days Of special interest is the report by Uryson and Kulaev (3) of growth (Table 2). A portion of the 15 liters of culture was that Propionibacterium freudenreichii has poly(P) glucoki- removed and the gas phase was restored with CO2 after each nase activity but only a trace of ATP glucokinase activity. removal to maintain anaerobic conditions. In the experiments of Tables 1 and 2, the packed cells were held at -70°C The publication costs of this article were defrayed in part by page charge until preparation of the crude extract. payment. This article must therefore be hereby marked 'advertisement" Crude Extracts and Fractionations. The cells were broken in accordance with 18 U.S.C. §1734 solely to indicate this fact. by passage through a French press or by grinding in an Ep- 312 Downloaded by guest on September 26, 2021 Biochemistry: Wood and Goss Proc. NatL. Acad. Sci. USA 82 (1985) 313 penbach mill (Gilford, Wood Company, Hudson, NY). For gether with ATP. With higher concentrations of EDTA, the the French press, 10-15 g of cells was suspended in twice the rate was high with ATP, and addition of poly(P) had no ef- volume of acetate, phosphate, or Hepes (Calbiochem) buffer fect. Apparently, poly(P) complexes an inhibitory metal, at 40C, containing 0.7 mM 2-mercaptoethanol/1.0 mM which is also complexed by EDTA. The reaction was also EDTA/0.1 mM phenylmethylsulfonyl fluoride. In some cas- investigated in the direction of synthesis of 1,3-diphospho- es, 10% (vol/vol) glycerol or 0.1 M glucose was included in glycerate and contained in ,umol, glycylglycine buffer (pH the buffer. After three passes through the French press at 7.2), 10; MgCl2, 3.9; NAD, 0.4; potassium phosphate buffer 8000 Pa, the mixture was centrifuged for 60 min at 16,000 x (pH 7.2), 5; fructose 1,6-P2, 0.4; ADP, 0.5; aldolase (Sigma), g, and the supernatant was collected as the crude extract. 0.8 units; glyceraldehyde-3-P dehydrogenase (Sigma), 2.7 For the Eppenbach mill, 250 g of cells was mixed with 150 ml units; and the extract. EDTA was not required and poly(P) of a similar buffer mixture together with 250 g of Pyrex was inactive. (iv) Pyruvate kinase: Assayed as described by beads, and the mixture was ground as described (9). To the Bucher and Pfleiderer (11). (v) Pyruvate, phosphate dikin- crude extract, an equal volume of an aqueous solution of ase: Assayed as described by Milner et al. (12). (vi) Carboxy- 10% (wt/vol) streptomycin was added at 40C with stirring, transphosphorylase: Assayed as described by Wood et al. and it was centrifuged after 20 min. The supernatant solution (13). (vii) Glycerol kinase: Assayed as described by Hayashi is designated the streptomycin-treated extract. This treat- and Lin (14). (viii) Poly(P) kinase: Assayed as described by ment removes nucleic acids and polyphosphates. The strep- Robinson et al. (5). (ix) Transcarboxylase: Assayed as de- tomycin-treated extract at 40C was brought to 35% satura- scribed by Wood et al. (9). tion with (NH4)2SO4. The precipitate was retained for deter- The enzyme activities are expressed as units per g of cells. mination of poly(P) kinase and the supernatant solution was For these calculations, it was assumed that the cells con- brought to 65% saturation; the resulting precipitate was dis- tained 80% water, and the total volume of the extraction flu- solved in 0.1 M phosphate (pH 6.5). id of the broken cell mixture was calculated to be 0.8 times Assay of Enzymes and Expression of Activity. All assays the weight in g of the cells broken plus the volume of buffer were done promptly if possible; when necessary, the ex- used in breaking the cells. tracts were stored at -70'C until assayed. Normally, the streptomycin-treated extract was used for the assays, but for RESULTS some enzymes the 35o-65% saturated (NH4)2SO4 fractions proved to give higher and more consistent values. The as- The yield of enzymes when the cells were grown on glucose says were done spectrophotometrically at -230C with two or and lactate are shown in Table 1. Those with glycerol as a more concentrations of extract to verify linearity with en- substrate have not been included, because they did not differ zyme concentration and were averaged for the calculation of significantly from the results with glucose or lactate except the units of activity (,umol/min). For tests of specificity of that glycerol kinase was somewhat increased. The enzyme the phosphate donors or acceptors, the usual acceptor or do- activities shown in Table 1 are derived from three separate nor was replaced with the phosphate compounds to be tested experiments. Of the three experiments, the highest and low- [ATP, PP,, and poly(P)3, poly(P)47, poly(P)200 from Sigma]. est activities have been averaged and the deviation of these The concentration of the replacing phosphate compound and two from this average is shown. There were substantial vari- of the Mg2+ were varied in the search for activity. ations in the activities of some of the enzymes. The varia- Methods of assay were as follows: (i) Poly(P) and ATP tions were not consistently correlated with the method or glucokinase: 300 ,u containing in ,umol, glucose, 2.3; MgCl2, buffer used in preparation of the extracts.

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