Kinetic Properties of Serratia Marcescens Adenosine

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Kinetic Properties of Serratia marcescens Adenosine 5'- Diphosphate Glucose Pyrophosphorylase JACK PREISS,* KATHLEEN CRAWFORD, JEANNE DOWNEY, CLAUDIA LAMMEL, AND ELAINE GREENBERG Department ofBiochemistry and Biophysics, University of California, Davis, California 95616 Received for publication 19 March 1976 The regulatory properties of partially purified adenosine 5'-diphosphate- (ADP) glucose pyrophosphorylase from two Serratia marcescens strains (ATCC 274 and ATCC 15365) have been studied. Slight or negligible activation by fructose-P2, pyridoxal-phosphate, or reduced nicotinamide adenine dinucleotide phosphate (NADPH) was observed. These compounds were previously shown to be potent activators of the ADPglucose pyrophosphorylases from the enterics, Salmonella typhimurium, Enterobacter aerogenes, Enterobacter cloacae, Citro- bacter freundii, Escherichia aurescens, Shigella dysenteriae, and Escherichia coli. Phosphoenolpyruvate stimulated the rate of ADPglucose synthesis catalyzed by Serratia ADPglucose pyrophosphorylase about 1.5- to 2-fold but did not affect the So.5 values (concentration of substrate required for 50% maximal stimulation) of the substrates, a-glucose-i-phosphate, and adenosine 5'-triphosphate. Adenosine 5'-monophosphate (AMP), a potent inhibitor of the enteric ADPglucose pyrophosphorylase, is an effective inhibitor of the S. marcescens enzyme. ADP also inhibits but is not as effective as AMP. Activators of the enteric enzyme counteract the inhibition caused by AMP. This is in contrast to what is observed for the S. marcescens enzyme. Neither phosphoenolpyruvate, fructose-diphosphate, pyridoxal-phosphate, NADPH, 3-phosphoglycerate, fructose-6-phosphate, nor pyruvate effect the inhibition caused by AMP. The properties of the S. marcescens HY strain and Serratia liquefaciens ADPglucose pyrophosphorylase were found to be similar to the above two S. marcescens enzynies with respect to activation and inhibition. These observations provide another example where the properties of an enzyme found in the genus Serratia have been found to be different from the properties of the same enzyme present in the enteric genera Escherichia, Salmonella, Shigella, Citrobacter, and En- terobacter. Previous reports (11, 19, 32, 33) have indi- The site of allosteric control of bacterial glyco- cated that adenosine 5'-diphosphate(ADP) glu- gen synthesis occurs at the enzymatic reaction cose is the glucosyl donor for the biosynthesis of where ADPglucose is synthesized. Glycolytic a-1,4-glucosidic linkages in bacterial glycogen intermediates stimulate the rate ofADPglucose via a reaction catalyzed by ADPglucose:1,4-a- synthesis, whereas compounds related to en- D-glucan-4- a-glucosyltransferase (bacterial ergy metabolism, adenosine 5'-monophosphate glycogen synthase, EC 2.4.21; reaction 1). (AMP), ADP, and Pi, inhibit ADPglucose synthesis (19, 32, 33). The nature of the activator ADPglucose + a-1,4-glucan = a-1,4-glucosyl- varied in each bacterial system and appears to glucan + ADP (1) be related to the carbon assimilation pathways The synthesis of ADPglucose was first dem- prevalent in the organism (11, 19, 32, 33). The onstrated in plants by Espada (9) and subse- ADPglucose pyrophosphorylase of several orga- quently in bacteria by Shen and Preiss (40) via nisms belonging to the Enterobacteriaceae were a reaction catalyzed by glucose-i-phosphate ad- found to be greatly stimulated by fructose-di- enylyltransferase (ADPglucose pyrophospho- phosphate, reduced nicotinamide adenine dinu- EC reaction cleotide phosphate (NADPH), and pyridoxal 5'- rylase, 2.7.7.27; 2). phosphate (35, 36). Metabolites such as 3-phos- adenosine 5'-triphosphate (ATP) + a-glucose-i- phoglyceraldehyde, phosphoenolpyruvate, and phosphate = ADPglucose + PPi (2) 2-phosphoglycerate gave significant but much 193 194 PREISS ET AL. J. BACTZRIOL. less activation. In contrast, preliminary ex- 1-butanol-pyridine-water (6:4:3); solvent D, 1-pro- periments showed ADPglucose pyrophospho- panol-ethyl acetate-water (7:1:2). The buffer system rylase present in crude extracts of Serratia used for high-voltage paper electrophoresis was 0.05 marcescens ATCC 15365 was not activated by M citrate buffer at pH 3.9. Assay of ADPglucose pyrophosphorylase. (i) Py- fructose-diphosphate (35). rophosphorolysis direction: assay A. Pyrophospho- This paper describes in more detail the ki- rolysis ofADPglucose was determined by the forma- netic properties of the ADPglucose pyrophos- tion of [32P]ATP from ADPglucose and 32pp1 (40). phorylases of some Serratia strains with re- The reaction mixture, which contained 20 ,umol of spect to their allosteric properties. Tris-hydrochloride buffer, pH 8.5, 2 gmol of MgCl2, 2.5 ,tmol of NaF, 100 gg of bovine plasma albumin, 0.5 ,umol of 32PP, (5 x 105 to 50 x 105 counts/min per MATERIALS AND METHODS ,umol), 0.3 ,umol of ADPglucose, activator when in- Bacterial strains. The following bacteria were dicated, and enzyme in a final volume of 0.25 ml, used: S. marcescens strains ATCC 15365, ATCC 274, was incubated at 37 C for 10 min. This assay was and ATCC 10759. These strains were obtained from used mainly to measure enzyme activity during the Department of Bacteriology, University of Cali- purification procedures. fornia, Davis. S. marcescens HY wild-type strain (ii) Synthesis direction: assay B. The synthesis of was obtained from W. L. Belser ofthe Department of ADPglucose was measured by following the forma- Biology of the University of California, Riverside. tion of ['4C]ADPglucose from [14C]glucose-l-phos- Serratia marinorubra ATCC 27614, Serratia liquefa- phate (12). The reaction mixture, which contained ciens ATCC 27592, and Serratia rubidea ATCC 27593 0.1 ,tmol of [14C]glucose-l-phosphate (5 x 10; to 9.0 were obtained from the American Type Culture Col- x 105 counts/min per ,umol), 10 ,umol of HEPES (N- lection Rockville, Md. All organisms were main- 2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) tained on nutrient agar slants and were grown in buffer, pH 8.0, 0.5 ,umol of ATP, 1.5 jAmol of MgCl2, liquid cultures that contained, in 1 liter: 11 mg of 100 ,ug of bovine plasma albumin, 1 IU of yeast CaCl2, 246 mg of MgSO4 7H20, 1.2 g of (NH4)2SO4, inorganic pyrophosphatase (Sigma), activator when 6.8 g of KH2PO4, 14.2 g of Na2HPO4, 1 ml of a trace indicated, and enzyme in a total volume of 0.2 ml, element solution (16), and 10 g of acid-hydrolyzed was incubated at 37 C for 10 min. When ADPglucose casein (Nutritional Biochemicals Corp.). The pH of synthesis was measured with the S. marcescens the media was adjusted to 7.2 with 1 M NaOH. D- ATCC 274 enzyme, 0.1 M HEPES, DH 7.5, was used Glucose at 0.6% (wt/vol), was added as a carbon as the buffer. source. S. marinorubra was also grown on minimal All kinetic studies were done in the range in media containing artificial seawater at 30 C (2). The which velocity of ATP or ADPglucose formation is media contained, in 1 liter: 0.1 M tris(hydroxy- proportional to protein concentration and time. methyl)aminomethane (Tris), pH 7.5, 1 g of NH4C1, Treatment of data to obtain kinetic constants. 0.25 g of FeSO4 * 7H2O, 0.057 g of K2HPO4, 11.7 g of Kinetic data are plotted as velocity versus substrate NaCl, 0.75 g of KCI, 12.35 g of MgSO4-7H2O, and or effector concentration and are replotted as a Hill 1.45 g of CaCl2 2H20. 1-Glucose at 0.75% (wt/vol), plot (5, 21, 41). The parameters determined from was added as a carbon source. The above cultures Hill plots are S0.5, A.5, or 1.5, corresponding to the were grown at 30 C in 2,800-ml Fernbach flasks on a concentration of substrate, activator or inhibitor re- New Brunswick rotary shaker for 16 h. quired for half-maximal velocity, activation, or in- The S. marcescens strains ATCC 274 and 15365 hibition, respectively (18), and n, the Hill constant. were also grown in a New Brunswick microfermen- Assay of glycogen synthase. Glycogen synthase tor in 10-liter cultures containing 1.1% K2HPO4, activity was assayed by measuring the rate of incor- 0.85% KH2PO4, and 0.6% yeast extract (Difco). The poration of labeled glucose into glycogen (34). The pH of the media was 7.0, and glucose at 1% (wt/vol) reaction mixture contained, in a volume of0.2 ml, 10 was added as a carbon source. The cultures were ,umol of Tris-chloride buffer, pH 8.0,,2 ,umol of glu- inoculated and aerated at 16 liters/min for 16 h at tathione (GSH), 5 jumol of KCI, 0.10 mg of bovine 30 C. The cells were harvested with a Sharples ul- plasma albumin, 1.0 ,umol of MgCl2, 0.5 mg ofrabbit tracentrifuge and stored as a paste at -12 C. liver glycogen, enzyme extract, and 0.1 ,Imol of Chemicals. [14C]glucose-phosphate was obtained ['4CIADPglucose (5 x 105 counts/min per ,Amol). from Amersham/Searle, England, and 32pp1 was ob- Assay of branching enzyme a-1,4-Glucan branch- tained from New England Nuclear Corp., Boston, ing enzyme (EC 2.4.1.18) was measured in uncentri- Mass. Diethylaminoethyl (DEAE)-cellulose was mi- fuged sonic extracts as previously described (15). crogranular preswollen DE-52 (Whatman). All other Partial purification of ADPglucose pyrophos- chemicals were obtained at the highest purity avail- phorylases S. marcescens ATCC 15365: disruption able from commercial sources. of cells. Crude extracts were prepared from 75 g of Methods. Protein determinations were done by bacterial cell paste in a Waring blender with glass the method of Lowry et al. (27). The following sys- beads as previously described (35). The. extracts tems were used for descending paper chromatogra- were centrifuged for 10 min at 30,000 x g, and the phy on Whatman no. 1 paper: solvent A, 95% supernatant fluid was used for further purification. ethanol-i M ammonium acetate, pH 3.8 (5:2); sol- All ensuing steps were done at 4 C. vent B, isobutyric acid-1 M NH3-0.1 M ethylenedia- Protamine sulfate precipitation. To 270 ml of the minetetraacetic acid, pH 7.2 (100:60:1.6); solvent C, crude supernatant was added, with slow stirring, 27 VOL. 127, 1976 S. MARCESCENS ADPGLUCOSE PYROPHOSPHORYLASE 195 ml of 1% protamine sulfate. After 10 min the suspen- lations for 5 min with a Biosonik III. The protamine sion was centrifuged for 15 min at 30,000 x g. step was omitted and the enzyme activity precipi- Ammonium sulfate fractionation. To 285 ml of tated between 0 and 0.4 saturated ammonium sul- the protamine sulfate supernatant fluid was added fate. A saturated ammonium sulfate solution was 190 ml of a saturated ammonium sulfate solution. used. The ammonium sulfate fraction was dialyzed After 10 min, the precipitate was centrifuged for 15 overnight against 2 liters of a 0.05 M HEPES buffer, min at 18,000 x g and the precipitate obtained was pH 7.0, which containined 1 mM dithiothreitol. The discarded. Solid ammonium sulfate was added to 450 dialyzed fraction was centrifuged for 1 h at 105,000 x ml of the supernatant fluid to give 80% saturation. g, and the supernatant fluid was adsorbed onto a The precipitate obtained after centrifugation (15 DEAE-cellulose column (2.4 by 11 cm) equilibrated min at 18,000 x g) was dissolved in 0.015 M pot,s- with 0.015 M potassium phosphate, pH 7.5. The sium phosphate buffer, pH 7.0, containing 5 mM enzyme was eluted with the same linear gradient dithiothreitol and dialyzed overnight against 500 ml used for the purification of the S. marcescens ATCC of the same phosphate buffer. 15365 enzyme. The fractions containing enzyme DEAE-cellulose chromatography. The ammo- were pooled, and solid ammonium sulfate was added nium sulfate fraction, 14 ml, was diluted to 28 ml to 0.7 saturation. After centrifugation for 15 min at with the above phosphate buffer and then adsorbed 30,000 x g, the precipitated enzyme was dissolved in on a DEAE-cellulose column (1.5 by 20 cm) equili- 0.05 M HEPES buffer, pH 7.0, containing 1.0 mM brated with 0.015 M phosphate buffer, pH 7.5. The dithiothreitol and dialyzed against 500 ml of the enzyme was eluted with a 1-liter gyadient composed same buffer overnight. of 500 ml of 0.015 M phosphate buffer solution, pH Table 1 summarizes the purification of both en- 7.5, containing 2 mM dithiothreitol in the mixing zymes. A unit ofactivity is defined as 1 jAmol ofATP chamber and 500 ml of 0.1 M phosphate buffer solu- formed from ADPglucose plus PP, in 10 min under tion, pH 7.0, containing 2 mM dithiothreitol and 0.3 the conditions of the pyrophosphorylase assay. The M KCI in the reservoir chamber. Enzyme activity DEAE-cellulose fraction for both enzymes had negli- appeared after 260 ml of the gradient had passed gible degrading activities towards ADPglucose, through the column and was collected in the next ATP, or pyrophosphate. No pyruvate kinase activity 100 ml of effluent. The fractions containing enzyme (EC 2.7.1.40) could be detected. A slight amount of activity were pooled, and solid ammonium sulfate phosphoglucomutase activity (EC 2.7.5.1), 0.011 was added to a concentration Of 0.85 saturation. ,tmol/min per mg of protein, was detected in the S. After 10 min, the precipitated enzyme was centri- marcescens ATCC 274 DEAE-cellulose fraction, and fuged for 10 min at 30,000 x g and dissolved in 0.03 no phosphoglucomutase activity was detected in the M phosphate buffer, pH 7.0, containing 5 mM dithio- strain ATCC 15365 DEAE-cellulose fraction. threitol. The enzyme solution was dialyzed over- Ultracentrifugation on sucrose density gradients night against the same buffer. (29). Linear sucrose gradients (4.3 ml) were pre- Partial purification of the S. marcewcens ATCC pared by mixing 5% (wt/vol) sucrose and 25% (wt/ 274 ADPglucose pyrophosphorylase. S. marcescens vol) sucrose solutions containing 0.05 M HEPES ATCC 274 was purified in the same manner as S. buffer, pH 7.0, and 1 mM dithiothreitol. Centrifuga- marcescens ATCC 15365, except the crude extracts tion was carried out as previously described (29) for were prepared by exposing 21 g of cell paste, sus- 16 h. Lactate dehydrogenase (EC 1.1.1.27) and pyru- pended in 100 ml of 0.1 M glycylglycine buffer, pH vate kinase were assayed as previously described (4, 7.0, containing 2.5 mM dithiothreitol, to sonic oscil- 17).

TABLE 1. Purification ofADPglucose pyrophosphorylases from S. marcescens strains ATCC 15365 and ATCC 274 Enzyme source Fraction Vol (ml) Total Protein Sp act Purification unifta (mg/ml) (units/mg) (fold) ATCC 15365 1. Crude extract 270 200 5.7 0.13 1 2. Protamine super- 285 190 3.0 0.22 1.7 natant 3. Ammonium sul- 14 102 25.6 0.29 2.2 fate 4. DEAE-cellulose 4.0 62 12.5 1.25 9.6 chromatography ATCC 274 1. Crude extract 100 211 15.4 0.14 1 2. Ammonium sul- 17 138 32 0.26 1.8 fate 3. 105,000 xg super- 14.8 152 28 0.37 2.6 natant fluid 4. DEAE-cellulose 5.5 96 6.1 2.87 21 chromatography a One unit is equal to 1 Amol of ATP formed in 10 min under the conditions specified for assay A in the absence of any activator. 196 PREISS ET AL. J. BACTERIOL. RESULTS TABLE 2. Nucleoside triphosphate specificity ofS. marcescens ADPglucose pyrophosphorylases" Properties of the purified ADPglucose pyrophosphorylases. (i) pH optima. The ADPglu- Nucleoside tri- ADPglucose formed (nmol) cose pyrophosphorylases from S. marcescens phosphate ATCC 274 ATCC 15365 strains ATCC 274 and 15365 showed optimal ADPglucose synthesis activity in HEPES None <0.05 0.12 buffer at pH 7.5 and 8.0, respectively. In Tris ATP 11.0 6.7 UTP 0.47 0.08 buffer the pH optimum was at 8.5 for both CTP 0.13 0.16 enzymes, and the activity was about 10 to 20% GTP <0.05 0.57 less than the activity observed in HEPES ITP <0.05 0.43 buffer at its optimal pH value. The following dATP <0.05 0.12 kinetic studies were, therefore, done in HEPES dTTP 0.23 buffer. ADPglucose synthesis activity was XTP 0.05 72% the about 50 and of pyrophosphorolysis "The standard synthesis assay (assay B) de- activity observed in the standard assay (see scribed in the text was used. The enzymes used were above) for strains ATCC 274 and ATCC 15365, the DEAE-cellulose fractions. Abbreviations: UTP, respectively. Uridine 5'-triphosphate; CTP, cytidine 5'-triphos- (ii) Nucleoside triphosphate specificity. Ta- phate; GTP, guanosine 5'-triphosphate; ITP, inosine ble 2 shows that the partially purified enzyme 5'-triphosphate; dATP, deoxyadenosine 5'-triphos- fractions were quite specific for ATP. Strain phate; dTTP, deoxythymidine 5'-triphosphate; XTP, ATCC 15365 enzyme showed slight activity xanthine 5'-triphosphate. with uridine 5'-triphosphate (4%), whereas strain ATCC 274 enzyme showed slight activity curve is hyperbolic with a Hill slope, n, of 0.99 with guanosine 5'-triphosphate (8.5%) and ino- ± 0.1 (three determinations) and a K5, of68 ± 9 sine 5'-triphosphate (6.4%). Omission of MgCl., ,uM. The ATCC 274 ADPglucose pyrophospho- in the assay resulted in virtually no ADPglu- rylase glucose-i-phosphate saturation curve cose synthesis. was also determined to be hyperbolic (Ft = 1.0) The radioactive product synthesized from with a Km for glucose-i-phosphate of 40 uM ATP and ['4C]glucose-1-phosphate co-chromat- (data not shown). ographed with ADPglucose in solvent systems In the pyrophosphorolysis direction the A and B and migrated with ADPglucose in ADPglucose saturation curve for the ATCC electrophoresis at pH 3.9. 15365 enzyme was found to be sigmoidal, with a (iii) Kinetic parameters. Figure 1 shows the Hill slope, n, of 1.7 and an So., of 0.32 mM. effect of ATP concentration on the velocity of Inhibition of S. marcescens ADPglucose ADPglucose synthesis catalyzed by strain pyrophosphorylase by 5'-AMP and ADP. Most ATCC 15365 ADPglucose pyrophosphorylase at bacterial ADPglucose pyrophosphorylases have two different MgCl, concentrations, 7.5 and been shown to be inhibited by AMP, ADP, or Pi 12.5 mM. Both curves are sigmoidal, giving (19, 32, 33). Figure 4 shows that S. marcescens Hill slope values, ni, of 1.67 ± 0.03 (three deter- ATCC 15365 enzyme is very sensitive to inhibi- minations) for 7.5 mM MgCl2 and 1.55 for 12.5 tion by AMP. The inhibition curve is essen- mM MgCl,. S0., values for ATP were 0.31 + 0.04 tially hyperbolic, and 50% inhibition (I.,) oc- mM for 7.5 mM MgCl2 and 0.29 mM for 12.5 mM curs at 15 A.M. ADP is also an effective inhibi- MgCl2. The SO., value of ATP for strain ATCC tor, with an Io., of 235 ,uM. Inorganic phosphate 274 ADPglucose pyrophosphorylase at 7.5 mM is a poor inhibitor, with an L0., of 8.7 mM. The MgCl, was 0.33 0.08 mM, and the Hill slope S. marcescens ATCC 274 enzyme was also in- value, n, was 1.97 + 0.15 (data not shown). hibited by AMP and had an lo., value of 31 1tM Figure 2 shows the effect of MgCl2 on the and a Hill constant, Ft, of 1.1. No inhibition by activity of the ATCC 15365 ADPglucose pyro- ADP was seen at 0.5 mM; at 1.5 mM ADP there phosphorylase. Maximal ADPglucose synthesis was 62% inhibition. No inhibition was observed was observed at 6.25 to 7.5 mM MgCl2. There with Pi up to 10 mM. were insufficient points to determine an So., Interaction of AMP with ATP. Figure 5 value for MgCl2 from Hill plots. The ATCC 274 shows that 4.8 uM AMP causes the ATP satu- enzyme also exhibited maximal activity at ration curve of the strain ATCC 15365 enzyme about 7.5 mM MgCl2 (data not shown). to become slightly more sigmoidal (Hill slope, Figure 3 shows the effect of glucose-i-phos- n, changes from 1.7 to 2.0) and increases the S0., phate on the activity of the ATCC 15365 ADP- value of ATP from 0.28 to 0.76 mM. At 0.2 mM glucose pyrophosphorylase. In contrast to the ATP, 4.8 ,uM AMP inhibited 88% of the activ- ATP saturation curve, the glucose-i-phosphate ity. This inhibition was partially overcome VOL. 127, 1976 S. MARCESCENS ADPGLUCOSE PYROPHOSPHORYLASE 197

0 0 E

C 0c E

(0 0 0 a-

ATP, mM

x 0 E

0 -J .05 0.1 0.2 0.5 1.0 2.0 3.0 ATP, mM FIG. 1. Effect ofATP concentration on ADPglucose synthesis catalyzed by the S. marcescens ATCC 15365 ADPglucose pyrophosphorylase. The conditions ofthe experiment were those ofassay B, except the concentration ofMgCl2 was 7.5 mM (0) or 12.5 mM (a), and the concentration ofATP was varied as indicated. (B) is a Hill plot of the data seen in (A). with increasing ATP concentrations, so that at most ADPglucose pyrophosphorylases have 2.5 mM the inhibition was only 20%. Moreover, been shown to be activated by glycolytic inter- the S. marcescens ATCC 15365 ADPglucose py- mediates (11, 19, 32, 33). Table 3 lists those rophosphorylase becomes more sensitive to compounds that have been shown to greatly AMP inhibition when the ATP concentration is activate ADPglucose pyrophosphorylases of decreased. The L.5 value decreased from 15 to many bacteria and plants. Very little activa- 2.7 aM as the ATP concentration was lowered tion (<20%) is seen with fructose-diphosphate, from 2.5 to 0.25 mM. The Hill constant, n, for fructose-6-phosphate, pyruvate, pyridoxal- the two AMP inhibition curves is 0.9, which is phosphate, and NADPH. Stimulations of 1.65- not appreciably different from the value of 1.0 fold with 2.0 mM phosphoenolpyruvate and for the AMP inhibition curve done in the pres- of 1.4-fold with 2.0 mM 3-phosphoglycerate ence of 2.5 mM ATP. Similar results were ob- were seen with the ATCC 15365 enzyme. The tained with the ATCC 274 enzyme; i.e., ATP ATCC 274 pyrophosphorylase was stimulated could partially relieve AMP inhibition. 1.4-fold by 2.0 mM phosphoenolpyruvate. The Activation of S. marcescens ADPglucose following compounds were tested at 2.0 mM and pyrophosphorylase. With one exception (37) stimulated the ATCC 15365 enzyme less than 198 PREISS ET AL. J. BACTERIOL. mate, L-alanine, L-aspartate, riboflavine 5'- n 20-_. .phosphate, NADH, and NADP. Ribose-5-phos- 0 phate (2.5 mM) and 2-keto, 3-deoxyphospho- E 6 \ gluconate (2 mM) gave 30% stimulation of ac- C 16-- ~ |tivity. Inhibition was observed with 2 mM cit- 6 rate (24%), 2 mM isocitrate (70%), 1.0 mM fla- Li 12- | \ vine adenine dinucleotide (58%), and NAD 0 (35%). Figure 6 shows the activation of ADP- glucose synthesis by varying concentrations of cnIL 8 phosphoenolpyruvate. Half-maximal activation 0 0 (A0,.,) was observed at 0.96 mM. The activation -J 4- l curve was hyperbolic in shape, giving a Hill constant (nf value) of 1.03. The AO.5 value of a.0D phosphoenolpyruvate for the S. marcescens ,,,,ATCC- 274 enzyme was determined to be 1.4 0 5 10 15 20 25 375 mM with a Hill constant (nf value) of 1.1. MgCI mM Phosphoenolpyruvate had no effect on the S,.5 X-I values of glucose-i-phosphate or ATP or on the FIG. 2. Effect ofMgCl2 concentration on ADPglu- cose synthesis catalyzed by the S. marcescens ATCC 100' 15365 ADPglucose pyrophosphorylase. The condi- .t A tions of the experiment were those of assay B, except the concentration ofMgCl2 was varied. 80- 60- 9 an z 40- L.J E 20- c a. -CV E 0 " 0 002 004 006 008 0 10 1 0 2 0 AMP, mM in 0 B u 80- 0C03 60- 025 Glucose-l-P, m M 40- z 1.2 rLJ B. 20- 1.0 0- - 08 L 06 02 04 06 08 0 12 14 25 1--~ ~ ~ ~ ~ ~ - ADP, mM 0.4 C 0.2 .0-- 0, / S-1~S' 100io 0 5- .i,. , ~~~50, . FIG. 3. Effect of glucose-1-phosphate concentra- E tion on ADPglucose synthesis catalyzed by the S. -0.5- _ 0 marcescens ATCC 15365 ADPglucose pyrophospho- -- .0. _ rylase. (B) is a reciprocal plot ofthe data seen in (A). The conditions of the experiment were those of assay 10 2 5 10 20 50 100 200 500 1000 B, except the concentration of glucose-i-phosphate INHIBITOR, jsMOLAR was varied. FIG. 4. Inhibition of the S. marcescens ATCC 10%; glucose-6-phosphate, 3-phosphoglyceral- 15365 ADPglucose pyrophosphorylase by AMP and dehyde, dihydroxyacetone-phosphate, glucose ADP. (C) is a Hill plot of the data in (A) and (B). Vma. is defined as activity in the absence ofinhibitor. 1,6-diphosphate, 2-deoxyribose-5-phosphate, The conditions of the experiment were those of assay acetyl coenzyme A, oxaloacetate, ca-keto-glu- B, except that AMP and ADP were added as indi- tarate, fumarate, succinate, malate, L-gluta- cated. VOL. 127, 1976 S. MARCESCENS ADPGLUCOSE PYROPHOSPHORYLASE 199

rylase activity are seen in the sonic extracts of 516 _ E S. liquefaciens and S. marcescens HY and

W 12. ATCC 15365 strains. Very minimal activities were observed for the pyrophosphorylase and cr 0 U. glycogen synthase in S. marcescens ATCC 8 11634, S. marinorubra, and S. rubidea ex- 0 tracts. Significant branching enzyme activity 4 was observed in all the extracts. a. The properties of the glycogen synthase in 0 04 0.8 1.2 1' 6 2.0 2.4 3.0 50 the crude extracts of S. marcescens strains HY, ATP, mM ATCC 274, and ATCC 15365 and of S. liquefa- FIG. 5. Effect ofAMP on the S. marcescens ATCC ciens were essentially the same. In the absence 15365 ADPglucose pyrophosphorylase ATP satura- of a glycogen primer, the ['4Clglucose transfer tion curve. Symbols: 0, no AMP added; *, 4.8 Am AMP added. The conditions of the experiment were those of assay B, except that AMP was added as 0 indicated and the ATP concentration was varied. E C TABLE 3. Activator specificity of the S. marcescens E Py voe ATCC 15365 ADPglucose 4 0 ATCC 274 and 0 - pyrophosphorylasesa PLei;o 4)~~~~. ADPglucose 0 Concn formed (nmol) 2 4 6 Activator (MM) A a. 15365 274 0 1.5 3.0 4.5 6.0 7.5 None 9.8 6.8 P-enol pyruv-te, mM Phosphoenolpyruvate 2.0 16.2 9.4 Fructose-diphosphate 1.5 10.7 7.4 FIG. 6. Activation of S. marcescens ATCC 15365 3-Phosphoglycerate 2.0 13.6 7.7 ADPglucose pyrophosphorylase by phosphoenolpyru- Fructose-6-phosphate 2.0 10.5 7.1 vate. Pyruvate 20 8.8 6.6 Pyridoxal-phosphate 0.5 11.1 7.1 TABLE 4. Levels ofglycogen biosynthetic enzymes in NADPH 1.0 8.6 7.0 Serratia extractsa 2-Phosphoglycerate 2.0 11.9 8.0 ADP- glucose a The conditions of the experiment were those of pyro GlyogenBranch- assay B, except activator was added at the concen- Protein pho- synthase ing en tration indicated. The enzymes used were the Organism (mg/ pho- (nmol/10 zyme DEAE-cellulose fractions. ml) rylase min/mg) (jsmol/ (nmoll 2 h/mg) 10 min/ 1.5 value of AMP for either the ATCC 15365 or mg) ATCC 274 pyrophosphorylases. The shape of S. marcescens 8.8 60 153 9.3 the substrate or inhibitor curves (Hill constant, HY n) also were not affected by phosphoenolpyru- S. marcescens 17.9 12.5 220 0.54 vate. It was also found that fructose-diphos- ATCC 274 S. marcescens 17.9 4 0.67 1.7 phate (2 mM), pyridoxal-phosphate (0.5 mM), ATCC 11634 fructose-6-phosphate (2 mM), NADPH (1 mM), S. marcescens 7.1 60 193 2.5 3-phosphoglycerate (2 mM), or pyruvate (20 ATCC 15365 mM) did not relieve any part of the 75% inhibi- S. marinoru- 14.3 5 2.7 0.71 bra tion caused by 50 AM AMP. Thus, the only S. liquefaciens 9.9 66 24.7 0.6 effect by phosphoenolpyruvate on the ADPglu- S. rubidea 19 2 2 1.0 cose pyrophosphorylases was on Vmax and not a The assays of ADPglucose pyrophosphorylase (assay on the apparent affinities of the substrates or A), glycogen synthase, and branching enzyme are described inhibitor, AMP. in the text. The enzyme extract was prepared by subjecting Presence of the glycogen biosynthetic en- a suspension of 1.5 g ofbacteria (wet weight) in 10 ml of 0.05 zymes in extracts of Serratia species. Table 4 M glycylglycine buffer, pH 7.0, containing 5 mM dithiothreitol to sonic oscillations for 1 to 3 min with a Biosonik III shows that the glycogen biosynthetic enzymes probe sonic oscillator. In the case of S. marcescens ATCC are present in a number of Serratia species. 15365, 3 g of bacteria was used. The assay of the various Equivalent levels of ADPglucose pyrophospho- enzymes was done with the uncentrifuged sonic extracts. 200 PREISS ET AL. J. BACTERIOL. was reduced to 10% or less of the rate observed in the presence of glycogen primer. Substitu- tion of uridine 5'-diphosphate glucose, thymi- E dine 5'-diphosphate glucose, cytidine 5'-diphos- c Lo phate glucose, guanosine 5'-diphosphate glu- 0 cose, or glucose-i-phosphate for ADPglucose in E 3 reaction mixtures resulted in less than 12% of 0 the activity observed for ADPglucose. Glycogen 0- synthase activity was not stimulated by the presence of either 0.5 mM pyridoxal-phosphate or 1.0 mM glucose-6-phosphate, fructose-6- phosphate, fructose-diphosphate, 3-phosphoglycerate, pyruvate, or phosphoenolpyruvate. 0 4 8 12 16 20 24 In each system the radioactive alcohol-insolu- FRACTION NO. ble products were hydrolyzed with ,8-amylase FIG. 7. Sucrose density gradient centrifugation of at pH 5.0, and in each case a radioactive mate- S. marcescens ATCC 274 ADPglucose pyrophospho- rial that co-chromatographed with maltose in rylase. Symbols: A, pyruvate kinase activity; 0, lac- solvent systems C and D was qbtained. This tate dehydrogenase activity; *, ADPglucose pyro- indicated that new a-1,4-glucosidic linkages phosphorylase activity using assay A. The procedure were being formed by transfer of [14C]glucose and enzyme assays are described in the text. The from ADPglucose to the primer glycogen. ordinate on the right side represents decrease in ab- inhibition of S. marcescens sorbance at 340 nm/min in the lactate dehydrogenase Activation and and pyruvate kinase assays. The left ordinate repre- HY and S. liquefaciens ADPglucose pyro- sents the ADPglucose pyrophosphorylase activity. phosphorylases. Table 5 shows that the ADP- glucose pyrophosphorylases of S. marcescens Pyridoxal-phosphate activated the HY strain strain HY and S. liquefaciens are also acti- enzyme about 30%, whereas 3-phosphoglycer- vated by phosphoenolpyruvate about 33 to 50%. ate activated the HY strain and S. liquefaciens 20 and None of TABLE 5. Effect ofmetabolites on S. marcescens HY enzymes 9%, respectively. and S. liquefaciens ADPglucose pyrophosphorylasea the other metabolites tested stimulated activity. Table 5 also shows that 50 ,uM AMP in- ADPglucose formed hibited the S. marcescens HY strain and S. (nmol) Activator Concn (mM) liquefaciens enzymes 43 and 37%, respectively. S. marces- S. liquefa- Greater inhibition is seen at 250 ,uM AMP. In cens HY ciens contrast, 0.5 mM ADP gave slight inhibition. None 5.4 5.4 Thus, the properties of the two ADPglucose Phosphoenol- 2.5 8.1 7.2 pyrophosphorylases appear to be quite similar pyruvate to those observed for the S. marcescens ATCC Pyridoxal- 0.5 7.0 4.8 274 and 15365 enzymes. phosphate Estimation of the molecular weight of the Fructose-di- 2.0 5.5 5.1 S. marcescens ATCC 274 ADPglucose pyrophosphate phosphorylase. The activity of the S. marces- Fructose-6- 2.0 5.6 4.2 phosphate cens ATCC 274 ADPglucose pyrophosphorylase Pyruvate 20 4.4 3.8 migrated as two peaks in sucrose gradient ul- 3-Phospho- 2.0 6.5 5.9 tracentrifugation (Fig. 7). One activity peak glycerate migrated slightly slower than rabbit muscle NADPH 2.0 5.2 4.6 pyruvate kinase, whereas the other migrated AMP 0.05 3.1 3.4 slower than rabbit muscle lactate dehydrogen- AMP 0.25 1.3 2.2 ase. The two activity peaks were reproducible ADP 0.5 4.5 5.0 in three separate ultracentrifugation runs. The a The enzymes used were prepared by fractiona- apparent molecular weights of peaks I and II tion of the sonic extracts (prepared as indicated in were determined to be 186,000 + 13,000 and Table 4) with a saturated ammonium sulfate solu- 96,000 + 10,000, respectively, using lactate de- tion at 4 C. The S. marcescens HY enzyme precipi- hydrogenase and pyruvate kinase as references tated between 0 to 0.4 saturation ammonium sulfate for molecular weights (140,000 and 237,000, re- and the S. liquefaciens enzyme precipitated between spectively). 0.4 and 0.6 saturation. Both enzymes were centrifuged at 30,000 x g for 10 min and redissolved in DISCUSSION 0.05 M HEPES buffer, pH 7.0, containing 2 mM dithioerythritol and were dialyzed overnight against The above results indicate that strains of the 100 volumes of the same buffer. genera Serratia contain an ADPglucose pyro- VOL. 127, 1976 S. MARCESCENS ADPGLUCOSE PYROPHOSPHORYLASE 201 phosphorylase distinct from those present in though no interaction between phosphoenolpy- other enterics with respect to the allosteric acti- ruvate and the inhibitor AMP is observed, the vator specificity (36). Whereas fructose-diphos- kinetic studies shown in Fig. 5 indicate interac- phate, NADPH, and pyridoxal-phosphate stim- tion between the substrate ATP and AMP. A ulates ADPglucose synthesis in extracts ofEn- decrease in the concentration of ATP increases terobacter cloacae, Enterobacter aerogenes, the sensitivity of the enzyme to AMP inhibi- Escherichia coli, Escherichia aurescens, Citro- tion, as indicated by a decrease in the 3.5. Like- bacter freundii, Salmonella typhimurium Lt-2, wise, the presence of AMP increases the SO, of and Shigella dysenteriae (unpublished data) 6- ATP (Fig. 5). Control of ADPglucose synthesis to 50-fold (36), very little or negligible activa- in Serratia would, therefore, appear to be regu- tion of ADPglucose synthesis in S. marcescens lated by energy charge, as has been determined and S. liquefaciens extracts is observed by the previously for E. coli B ADPglucose pyrophos- above three metabolites. Optimal activation phorylase (19, 33, 39). However, modulation of was observed with phosphoenolpyruvate and the energy charge control of ADPglucose pyro- was only 1.6-fold. In all systems where signifi- phosphorylase activity by glycolytic intermedi- cant allosteric activation of ADPglucose pyro- ates appears to be lacking in Serratia. phosphorylase has been studied (33), the activa- Figure 7 shows that S. marcescens ADPglu- tor has been shown to increase the apparent cose pyrophosphorylase activity migrated as affinity of the enzyme for the substrates ATP two peaks in sucrose density ultracentrifuga- and glucose-i-phosphate (i.e., So., of the sub- tion experiments, with apparent molecular strates is lowered in the presence of activator). weights of 96,000 and 186,000. This is in con- In contrast, phosphoenolpyruvate does not af- trast to the observation of only one activity fect the So.5 values of glucose-l-phosphate or peak for the E. coli B enzyme, which had a ATP for the S. marcescens ATCC 15365 or molecular weight of about 200,000 (14, 31). The ATCC 274 ADPglucose pyrophosphorylases. native E. coli B enzyme has been shown to The S,.5 values of ATP and glucose-i-phos- consist of four subunits, with identical molecu- phate, however, are essentially the same as lar weights of 50,000 (14). The subunit molecu- those observed for the other enteric ADPglu- lar weight ofthe S. marcescens enzyme may be cose pyrophosphorylases in the presence of similar to that of the E. coli enzyme. If so, the their activators (33). The low activation by S. marcescens enzyme probably exists as tetra- phosphoenolypruvate occurs only at moderately meric and dimeric forms in equilibrium with high concentrations when compared with acti- each other. vators ofother ADPglucose pyrophosphorylases Studies on the immunological cross-reactivi- (33). Half-maximal activation (AO.5) of the S. ties of the a and 12 subunits of tryptophan marcescens enzymes occurred at 0.96 to 1.4 mM synthase from representative species of various phosphoenolpyruvate. Whereas the concentra- genera of the enteric bacteria (7, 30, 38) and on tion of phosphoenolpyruvate in S. marcescens the immunological relationships of the alkaline cells has not been reported, the phosphoenol- phosphatases from the same species (6) show pyruvate concentration in glucose-minimal me- that the enzymes from Serratia have less cross- dium-grown E. coli K-12 cells in exponential reactivity with the antiserum prepared against phase has been determined to be 0.088 mM (26). the E. coli protein than the corresponding en- In succinate-grown cells the concentration was zymes from the Escherichia, Shigella, Salmo- 0.96 mM (26). Phosphoenolpyruvate does not nella, Citrobacter, and KlebsiellalEnterobacter have any effect on the inhibition caused by group. Furthermore, partial and full primary AMP. Phosphoenolpyruvate activation of S. sequence analysis of the a chain of the trypto- marcescens ADPglucose pyrophosphorylase in phan synthase indicates that the S. marcescens vivo, therefore, does not appear to be signifi- protein is more dissimilar in sequence to the E. cant. coli a chain than are the S. typhimurium, E. AMP is a potent inhibitor of the S. marces- aerogenes, and S. dysenteriae a chains (7, 13, cens and S. liquefaciens ADPglucose pyrophos- 22-25). phorylases, and in this respect they are similar In a study of other enzyme activities in the to the ADPglucose pyrophosphorylases of E. tryptophan biosynthetic pathway, it was ob- coli B (33, 35), S. typhimurium, and C. freundii served that the anthanilate synthase (EC (36). ADP and Pi are much less effective inhibi- 4.1.3.27) and anthranilate phosphoribosyl tors. The AMP and ADP inhibition curves of transferase (EC 2.4.2.18) activities reside in a the S. marcescens enzyme are hyperbolic, in single large enzyme complex in E. coli, Salmo- contrast to what is observed with the ADPglu- nella sp., and E. aerogenes (7, 20). In S. mar- cose pyrophosphorylases from E. coli B (33, 35), cescens, S. marinorubra, and Enterobacter li- S. typhimurium, and C. freundii (36). Al- quefaciens (now classified as Serratia liquefa- 202 PREISS ET AL. J. BACTERIOL. ciens [1, 8, 10]), the two activities reside in liquefaciens (Gimes and Hennerty) Bascomb et al. (formerly Enterobacter liquefaciens) and Serratia separate polypeptides (20). rubidaea (Stapp) comb. nov. and designation of type All the above studies, as well as in vitro and neotype strains. Int. J. Syst. Bacteriol. 23:217- deoxyribonucleic acid hybridization studies (3, 225. 28), clearly show the close macromolecular sim- 11. Furlong, C. E., and J. Preiss. 1969. Biosynthesis of bacterial glycogen. VII. Purification and properties of ilarities of the Escherichia, Shigella, Salmo- the adenosine diphosphoglucose pyrophosphorylase of nella, Citrobacter, and Enterobacter/Klebsiella Rhodospirillium rubrum. J. Biol. Chem. 244:2538- genera. The dissimilarities of the genus Serra- 2548. tia to genera ofthe above enteric bacteria in the 12. Ghosh, H. P., and J. Preiss. 1966. Adenosine diphosphate glucose pyrophosphorylase. A regulatory en- numerous protein and deoxyribonucleic acid zyme in the biosynthesis of starch in spinach leaf hybridization studies mentioned have caused chloroplasts. J. Biol. Chem. 241:4491-4504. some investigators to hypothesize that Serratia 13. Guest, J. R., G. R. Drapeau, B. C. Carlton, and C. diverged earlier from a common ancestor of the Yanofsky. 1967. The amino acid sequence of the A protein (a subunit) of the tryptophan synthetase of enterobacteria than the above genera (6, 20). Escherichia coli. V. Order of tryptic peptides and the The finding of an ADPglucose pyrophosphoryl- complete amino acid sequence. J. Biol. Chem. ase in Serratia with different allosteric proper- 242:5442-5446. ties than in the Escherichia, Shigella, Salmo- 14. Haugen, T., A. Ishaque, A. K. Chatterjee, and J. Preiss. 1974. Purification ofEscherichia coli ADPglu- nella, Citrobacter, and EnterobacterlKlebsiella cose pyrophosphorylase by affinity chromatography. genera is consistent with this view. The finding Fed. Eur. Biochem. Soc. Lett. 42:205-208. that S. liquefaciens ADPglucose pyrophospho- 15. Hawker, J. S., J. L. Ozbun, H. Ozaki, E. Greenberg, rylase has very similar allosteric properties to and J. Preiss. 1974. Interaction of spinach leaf adenosine diphosphate glucose a-1,4-glucan a-4-glucosyl the S. marcescens enzyme and different proper- transferase and a-1 ,4-glucan, a-1,4-glucan-6-glucosyl ties than those found for E. aerogenes and E. transferase in synthesis of branched a-glucan. 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