Phosphorylation Enzymes of the Propionic Acid Bacteria and The

Home , Fructokinase, Glucokinase, Glycerol kinase, Mannokinase, Phosphofructokinase, Phosphoglycerate kinase

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 species. 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. Many of the ex- 2.3; Tris HCI (pH 7.5), 46; NADP, 0.15; glucose-6-phos- tracts were prepared with acetate buffer (0.25 M, pH 5.5), phate dehydrogenase (Sigma), 0.3 units; and the extract. The but a variety of tests with 50 mM Hepes (pH 7) and 0.25 M reaction was initiated by addition of 5 A1l of 0.05 M poly(P)200 phosphate buffer (pH 6.5) did not significantly reduce this or 5 ,ul of 100 mM ATP. (ii) PP1 and ATP phosphofructoki- variation nor did the inclusion of glucose or glycerol in the nase: Assayed as described by O'Brien et al. (10). (iii) 3- buffer. Glucose was included because it aids in stabilizing Phosphoglycerate kinase: 300 ,ul containing in ,umol, poly(P) glucokinase (15), and glycerol was added in others, Tris-HCI (pH 6.9), 30; MgCl2, 1.5; EDTA, 25; cysteine, 0.9; because Smart and Pritchard (16) found that it stabilizes py- ATP, 0.25; NADH, 0.1; 3-phosphoglycerate (Sigma; sodium ruvate kinase. We observed, as reported by Smart and salt), 5; and glyceraldehyde-3-P dehydrogenase (Sigma), 1.4 Pritchard (16), that there was a lag of as much as 6-10 min units. With lower concentrations of EDTA, there was no ac- before the maximum rate of the pyruvate kinase reaction tivity with poly(P), but there was considerable activity with was attained. The maximum rate was used for calculation of ATP, which was much increased if poly(P) was added to- units.

Table 1. Enzyme content of three species of propionic acid bacteria grown on glucose or lactate Lactate as substrate Glucose as substrate P. freuden- P. arab- P. sher- P. freuden- P. arab- P. sher- Enzyme reichii inosum manii reichii inosum manii Poly(P) glucokinase 8.6 + 2.4 0.6 ± 0.4 5.2 ± 1.5 5.6 + 2.1 0.5 ± 0.5 7.1 ± 1.5 ATP glucokinase 0.9 ± 0.4 0.9 + 0.3 1.3 ± 0.1 1.2 + 0.4 0.3 ± 0.1 2.3 ± 0.6 PPj phosphofructokinase 86 ± 18 44 + 30 96 ± 18 46 ± 4 53 + 35 44 + 18 ATP phosphofructokinase 0.15 ± 0.15 0.0 ± 0.0 3.9 ± 1.7 1.0 ± 0.8 0.1 + 0.1 0.5 ± 0.1 ATP phosphoglycerate kinase 19 ± 7 20 ± 1 32 + 20 2.9 ± 1.0 18 ± 4 30 + 16 Pyruvate kinase 4.0 ± 1.7 15 ± 6.0 7.7 ± 4.3 5.4 + 1.0 23 ± 19 6.6 ± 4.4 Pyruvate phosphate dikinase 0.9 ± 0.4 0.6 ± 0.2 3.8 ± 2.5 1.8 + 1.2 2.7 ± 2.4 2.6 ± 1.9 Carboxytransphosphorylase 6.2 ± 3.0 0.6 ± 0.0 6.0 ± 4.0 1.5 ± 1.1 0.3 ± 0.1 6.1 ± 0.3 Glycerol kinase 18 ± 2.5 16 ± 1.5 5.6 ± 0.5 3.3 ± 1.1 8.7 + 3.3 3.1 ± 0.7 Poly(P) kinase ND ND 0.3 + 0.1 0.3 ± 0.1 ND 0.2 ± 0.1 Transcarboxylase 132 + 14 146 + 73 217 ± 6 43 ± 13 58 ± 12 137 + 34 Enzyme content indicated as units/g (,umol of product formed per min by the enzyme from 1 g of cells). ND, not determined. Downloaded by guest on September 26, 2021 314 Biochemistry: Wood and Goss Proc. NatL Acad Sci. USA 82 (1985)

Table 2. Enzyme content of three species of propionic acid bacteria at different phases of growth with lactate as substrate P. shermanii P. arabinosum P. freudenreichii 49 hr 91 hr 164 hr 53 hr 97 hr 169 hr 48 hr 96 hr 171 hr Enzyme (2.8) (4.6) (4.4) (2.2) (3.0) (3.9) (3.1) (5.3) (6.0) Poly(P) glucokinase 3.5 5.5 5.9 0.13 0.08 0.33 4.7 7.7 6.3 ATP glucokinase 0.53 0.75 1.0 0.53 0.60 0.54 0.81 1.2 1.2 PPj phosphofructokinase 80 60 58 47 36 20 78 61 45 ATP phosphofructokinase 1.1 1.3 1.9 0.26 0.17 0.18 2.5 1.1 0.81 3-Phosphoglycerate kinase 32 37 36 27 45 28 48 51 47 Pyruvate kinase 4.8 5.0 3.3 33 13 16 3.7 3.3 3.1 Pyruvate, phosphate dikinase 5.3 4.2 6.1 4.0 1.3 0.72 3.7 3.8 3.5 Carboxytransphosphorylase 2.2 4.4 4.7 0.53 0.81 0.62 5.5 11 10 Glycerol kinase 4.9 6.7 9.7 25 26 26 32 31 23 Poly(P) kinase 0.06 0.20 0.14 0.09 0.08 0.08 0.11 0.16 0.14 Transcarboxylase 81 104 112 3.3 19 10 97 113 110 mg of protein per g of cells 66 71 62 20 27 19 67 64 53 Extracts were prepared using a French press. Enzyme content indicated as units/g (/Lmol of product formed per min by enzyme from 1 g of cells). Numbers in parentheses represent g of cells per liter.

It was considered that the enzyme activity might change at if the activities of both enzymes are added together, the rates different stages of growth and be the cause of the variation. are not sufficient to account for the utilization of 1.8 ,umol of To investigate this possibility, the three species were grown glucose per g of cells per min. It is noted that the yield of on lactate and harvested in the logarithmic phase of growth other enzymes was not uniformly low in P. arabinosum (2 days), when approaching the stationary phase (4 days), grown on glucose. The glucokinase reaction was tested with and during the stationary phase (7 days). The results are P-enolpyruvate, GTP, and ITP in place of ATP, but no sig- shown in Table 2. There was some variation in the enzyme nificant activities were observed. Perhaps the glucokinase(s) content; the most consistent being of PP1 phosphofructoki- remains membrane bound with this species. nase, which decreased with time in all three species, and of Based on the report of Uryson and Kulaev (3), we consid- transcarboxylase, which increased from the logarithmic to ered that the activity of ATP glucokinase with P. freudenrei- the stationary phase. However, the variations observed in the chii might be far below 1.8 units per g of cells and thus pro- experiments of Table 2 do not account for those of Table 1. vide evidence that poly(P) glucokinase is required in addition For determination of the rate of glucose utilization, the to ATP glucokinase. Our observed activity was low but not amount (,tmol) of glucose fermented during a 2-day interval significantly <1.8. However, with P. shermanii and P. freu- of the stationary phase of growth was determined when the denreichii, poly(P) glucokinase was substantially more ac- cell density was nearly constant. With P. arabinosum, 93.7 tive than ATP glucokinase, and there is every reason to con- mmol of glucose was fermented per liter and the wet weight sider that poly(P) is used by these species to phosphorylate of cells was 17.1 g. Thus, the ,umol of glucose fermented per glucose. It is noteworthy that Szymona and Szumilo (17) min was 93,700/(48 x 60) = 32.5 and the umol-min-l g-1 found when Mycobacterium phlei was grown on fructose was 32.5/17.1 = 1.9. With P. freudenreichii, the correspond- that poly(P) fructokinase was present, but it was absent from ing values were 55.1 mmol ofglucose fermented and 11.1 g of the cells grown on glucose. Two other adaptive enzymes cells; thus, the ,mol min-1 g-1 was 1.7. With P. shermandi, were discovered (18): poly(P) mannokinase and poly(P) glu- the values were 78 and 14.3, giving 1.9 ,mol min-l g-1. For conatokinase. It seems unlikely there would be such adapta- the present purposes, a value of 1.8 ,umol ofglucose ferment- tion if the poly(P) were not actually being utilized. ed per min per g of cells was used for all three species. PP; Phosphofructokinase. The activity of PP, phosphofructokinase was high with all three species and with all three DISCUSSION substrates for growth, whereas that of ATP phosphofructokinase was low. Fructose 2,6-P2 activates ATP phosphofruc- Rationale of Interpretation of the Data. Under anaerobic tokinase from animals (19) and PP1 phosphofructokinase conditions, the propionic acid bacteria ferment glucose by from plants (20), but it had no effect when added to our phos- the Embden-Meyerhof pathway. From the reactions of this phofructokinase assays. It appears from these results that pathway, the minimum activity of the respective enzymes PP1 phosphofructokinase is the major catalytic agent for for- necessary to meet the requirements of the observed rate of mation of fructose 1,6-P2. glucose metabolism can be predicted. Since 1 ,mol of glu- In contrast to the report of Bertagnolli and Cook (6), both cose 6-P and fructose 1,6-P2 are formed per ,umol of glucose poly(P)3 and poly(P)4 were found to be inactive in the phos- fermented, the glucokinase and phosphofructokinase activi- phofructokinase reaction. They did promote an initial forma- ty per g of cells per min (units/g in Table 1) should be at least tion of fructose 1,6-P2 but the rate decreased with time. Ex- 1.8, and if 2 mol of an intermediate compound is formed per amination of the poly(P)3 and poly(P)4 by chromatography as mol of glucose, the units/g for the enzyme catalyzing the described by Shigern and Van Wazer (21) showed that the formation of these intermediates should be 3.6 or greater. poly(P)3 contained a small amount of PP, and the poly(P)4, Poly(P) and ATP Glucokinase. Glucose was phosphorylat- some poly(P)3, PP;, and Pi. It is concluded the activity with ed both by ATP and by poly(P) and with both P. shermanii poly(P)3 and poly(P)4 (Sigma) was due to their contamination and P. freudenreichii, the rate of phosphorylation was con- by P1 and PPi. With poly(P)4, there was some formation of sistently more rapid with poly(P) than with ATP. With P. fructose 6-P from fructose 1,6-P2 apparently because of its arabinosum, a low rate of phosphorylation was observed contamination by Pi. We have been informed by B. F. Cook with both donors and sometimes phosphorylation by ATP (personal communication) that they no longer find poly(P) was greater than with poly(P). There was no activity with PPj active in the phosphofructokinase reaction. in this reaction with any of the three species. The low activi- ATP 3-Phosphoglycerate Kinase. The activity clearly was ty with ATP or poly(P) with P. arabinosum is puzzling. Even adequate to meet the requirement of the glycolytic pathway. Downloaded by guest on September 26, 2021 Biochemistry: Wood and Goss Proc. Natl. Acad. Sci. USA 82 (1985) 315

The activities shown in Table 1 are for the conversion of 3- more active than we have observed. Until proven otherwise, phosphoglycerate to 1,3-diphosphoglycerate. In glycolysis, we consider the following to be true. A significant amount of 1,3-phosphoglycerate is converted to 3-phosphoglycerate poly(P) is not generated via the phosphoglycerate kinase re- and the activity has been reported (22) to be 4 times faster in action. Poly(P) is not utilized in the formation of fructose this direction than in the reverse direction. We usually ob- 1,6-P2. Aside from those reactions catalyzed by glucokinase, served even greater differences. With P. freudenreichii, a there are no other reactions of the glycolytic pathway of pro- value of 2.9 was observed (Table 1), but in the forward direc- pionic acid bacteria that utilize more than one phosphate do- tion the value was much greater than 3.6 units/g. We ob- nor or acceptor. served no activity with poly(P) or PP1 as either donors or acceptors. The activity with poly(P) reported by Kulaev et We thank Nancy Robinson for assaying the polyphosphate ki- al. (4) (see also ref. 23) was extremely low, the highest being nase. This investigation was supported by Public Health Service 0.6 x 10-3 units per mg of protein for propionic acid bacte- Grant GM 29569 from the National Institutes of Health and by Grant ria. It, therefore, is doubtful that poly(P) or PP, have a signif- PCM 8022701 from the National Science Foundation. icant role in this reaction. Pyruvate Kinase and Pyruvate, Phosphate Dikinase. For- mation of pyruvate from P-enolpyruvate can be catalyzed by 1. Wood, H. G. (1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36, either pyruvate kinase or by pyruvate, phosphate dikinase 2197-2205. in all pyruvate is via pyruvate kinase 2. Wood, H. G., O'Brien, W. E. & Michaels, G. (1977) Adv. En- but probability, formed zymol. 45, 85-155. (2). When the bacteria are grown on lactate or pyruvate, P- 3. Uryson, S. 0. & Kulaev, 1. S. (1968) Dokl. Biol. Sci. (Engi. enolpyruvate must be synthesized for use in anabolic reac- Transl.) 183, 697-699. tions and pyruvate, phosphate dikinase via Reaction 2 (right 4. Kulaev, I. S., Vorob'eva, L. I., Konovalova, L. V., Bobyk, to left) serves this purpose. In the fermentation of glucose, M. A., Konoshenko, G. I. & Uryson, S. 0. (1973) Biochemis- there is no obvious requirement for pyruvate, phosphate di- try (Engi. Transl.) 38, 595-599. kinase. It could provide a mechanism of converting ATP to 5. Robinson, N., Goss, N. H. & Wood, H. G. (1984) Biochem. PP, by reversal of Reaction 2, but this seems unlikely. No Int. 8, 757-769. activity was observed with poly(P) in this reaction. Wide 6. Bertagnolli, B. & Cook, B. F. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, 2076 (abstr.). fluctuations in the activity of pyruvate kinase were ob- 7. Wood, H. G. (1982) in Oxygen Fuels and Living Matter, Part served, but in most fermentations the activity was >3.6 un- 2, ed. Semenza, G. (Wiley, New York), pp. 173-250. its/g. There is activation of the enzyme with time (16), which 8. Virtanen, A. I. (1923) Soc. Sci. Fenn. Commentat. Phys- may be influenced by unknown factors and cause the ob- Math. 1, 1-23. served variations. Poly(P), PPi, or Pi did not function as 9. Wood, H. G., Jacobson, B., Gerwin, B. I. & Northrop, D. B. phosphate acceptors in this reaction. (1968) Methods Enzymol. 13, 215-231. Carboxytransphosphorylase. This reaction is the source of 10. O'Brien, W. E., Bowien, S. & Wood, H. G. (1975) J. Biol. the C4-dicarboxylic acids for anabolic reactions and for for- Chem. 250, 8690-8695. mation of succinate when it is an end product of the fermen- 11. Bucher, T. & Pfleiderer, G. (1955) Methods Enzymol. 1, 435- 440. tation. Oxalacetate also can be formed from pyruvate by 12. Milner, Y., Michaels, G. & Wood, H. G. (1975) Methods Enzy- transcarboxylation from methylmalonyl-CoA but there is no mol. 42, 199-212. net increase of C4-dicarboxylic acids by this sequence. The 13. Wood, H. G., Davis, J. J. & Willard, J. M. (1969) Methods En- reaction is specific for PP, and Pi; there was no activity with zymol. 13, 297-309. poly(P) or ATP. 14. Hayashi, S. & Lin, E. C. C. (1967) J. Biol. Chem. 242, 1030- Polyphosphate Kinase. This is the only enzyme we have 1035. found, thus far, in the propionic acid bacteria that catalyzes 15. Szymona, 0. & Szymona, M. (1979) Acta Microbiol. Pol. 28, the synthesis of poly(P). The largest value shown in Table 1 153-160. 16. Smart, J. B. & Pritchard, G. G. (1979) Arch. Microbiol. 122, is 0.3 ± 0.1 units per g of cells, which would be insufficient if it occurred 281-288. for the requirement of glucose 6-P synthesis, 17. Szymona, 0. & Szumilo, T. (1966) Acta Biochim. Pol. 17, 129- only via poly(P) glucokinase. The possibility that poly(P) 143. may be synthesized via an electron transport membrane 18. Szymona, O., Kowalska, H. & Szymona, M. (1969) Ann. bound system has been proposed, and the relevant literature Univ. Mariae Curie-Sklodowska Sect. D 24, 1-18. has been reviewed (23, 24). 19. Hers, H.-G. & Van Schaftingen, E. (1982) Biochem. J. 206, 1- Glycerol Kinase. It was found to be specific for ATP. 12. There was no reaction with poly(P) or PPj. 20. Sabularse, D. C. & Anderson, R. L. (1981) Biochem. Biophys. Transcarboxylase. It was found to be abundant in each of Res. Commun. 103, 848-855. R. Anal. Chem. 1984- the three species grown on the three substrates and is in ac- 21. Shigern, 0. & Van Wazer, J. (1963) 35, the reaction 1985. cord with the view that this enzyme catalyzes by 22. Krietsch, W. K. G. & Bucher, T. (1970) Eur. J. Biochem. 7, which propionate is formed in propionic acid bacteria. Previ- 568-580. ously, this enzyme had not been demonstrated in species 23. Kulaev, I. S. (1979) The Biochemistry ofInorganic Polyphos- other than P. shermanii. phates, Engl. transl. (Wiley, New York). Concluding Comments. It is always possible that condi- 24. Kulaev, I. S. & Vagabov, J. M. (1983) Adv. Microbiol. Physi- tions may ultimately be found in which an enzyme will be ol. 24, 83-171. Downloaded by guest on September 26, 2021