Izui, Katsura

Katsuki, Hirohiko

Teraoka, Hirobumi

English

Kyoto University Research Information Repository

RIGHT:URL:CITATION:AUTHOR(S):ISSUE DATE:TITLE:Partial Purification and Propertiesof PhosphoenolpyruvateCarboxykinase of Escherichia ColiWTeraoka, Hirobumi; Izui, Katsura; Katsuki, HirohikoTeraoka, Hirobumi ...[et al]. Partial Purification and Properties of Phosphoenolpyruvate Carboxykinase of EscherichiaColi W. Memoirs of the Faculty of Science, Kyoto University. Series of physics, astrophysics, geophysics and chemistry1977, 35(1): 141-1471977-09http://hdl.handle.net/2433/257562Memoirs of the Faculty of Science, Kyoto University, Series of Physics, Astrophysics,        Geophysics and Chemistry, Vol. XXXV, No. 1, Article 9, 1977.PARTIAL PURIFICATION AND PROPERTIES OFPHOSPHOENOLPYRUVATE CARBOXYKIN ASE            OF ESCHERICHIA COLI W               ByHirebumi TERAOKA*, Katsura IZUI               and        Hirehiko KATSUKIDepartment of Chemistry, Faculty of Science, Kyoto University, Kyoto               (Receiyed September 2, 1975)                           SUMMARY   PIiosphoenolpyruvate carboxykinase was purified 85-fold in a yield of 24e/, overcetl-free extract of Eschericltia coli W. The molecular weight was estimated to be64,eOO by the method of gel fiItration.   WheR assayed in a direction of C02-fixation, the enzyme showed a pH optimumat 7.0 and required MR2" for the activity (Km =O.6 mM). Km values for phosphoenol-pyruvate, ADP and KHC03 were O.6, O.4 and 40 mM, respectively.   When assayed in a direction of decarboxylation, Km value for oxaloacetate wasO.6 mM. The plots of reaction velocity against ATP concentration showed a character-istic curve for substrate inhibition at higher concentratioRs thaR 1 mM. Km valuefor ATP was about O.2 mM.   The enzyme activity was not affected by tke metabolites tested, including inter-mediates of tlie glycolytic pathway and the tricarboxylic acid cycle, and pyridinenucleotides.Introduction    Phosphoenolpyruvate carboxykinase [EC 4.1.1.32] which catalyzes PEP**-formation reaction froin oxaloacetate and ATP is known to be a key enzyme ingluconeogenesis (1). Recent studies on the levels of the enzyme of Escherichiaeoli grown under various nutritional conditions by }lsie and Rickenberg (2), Shragoand Shug (3), and by us (4) indicate that the level of the enzyme is high in gluconeo-genesis and low in glycolysis. Wright and Sanwal (5), using two assay methods,i.e. isotope exchange assay and Ce2-fixation assay, reported that the partially pu-rified enzyme from E. eoli B is inhibited by NADH in an allosteric manner.    In this communication, a partial purification of PEP carboxykinase froin E.coli W and kinetic properties of the enzyme in the reactions of both decarboxylation * Present address: Department of I'athological Biochemistry, Medical Research Institute,  Tokyo IV!edlca! and Dental University, Tokyo.** Abbreviations: PEP, phosphoenolpyravate; DTT, dithiotkreitol; Tris, tris (hydroxylmethyl)-  aminomethane.142 H. TERAOKA, K. IZUI aRd H. KATSUKIand C02-fixation directions are described. Regulatory mechanism of the enzyme isalso discussed.                   MATERIALS AND METHODSChemicals   Malate dehydrogenase [EC 1.1.l.37] was obtained frofn Boehringer-Mannheim.DTT, Tris, bovine serum albumin, NADH, NAD" and Fast violet B were Sigmaproducts. Sephadex G-100 and DEAE-Sepahdex were obtained from Pharmacia.PEP was prepared by the inethod described previously (6). All other chemicalswere purchased from Nakarai Chemicals Co. (Kyoto).Preparation of Cell-Nfree Extract   E. coli W was grown aerobically with shaking at 300C in a medium containing10 g ofpolypeptone, 10 g of yeast extract and 5 g of K,HPO, in 1 1 of distilled water.Tlie cells were harvested near tke end of the logarithmic growth phase and washedtwice with 10/, KCI solution. The cells suspended in O.l M Tris-HCI btffer, pH 7.5,containing 1 rnM EDTA and O.1 mM DTT (O.5 g wet cellslml) were disrupted witha Ohtake sonic disintegrator at 20 kHz for 10 min at 4eC. This suspension wascentrifuged at 25,OOOÅ~g for 40 min and the resulting supernatant was used as theenzyme source for purification.Enzyme Assays   1. Assay of the enzyme iR the directien of Ce2-fixation-Tke standard reac-tion mixture contained 5 Jtmoles of PEP, 5 ptmoles of ADP, 10 pemoles of MnS04,50 ptmoles of KHCO,, O.15 ptmole of NADH, 1 l.U. of malate debydrogenase,O.2 ltmole of DTT and 70 pamoles of Tris-inaleate buffer, pH 7.0, in a total volumeof 1.0ml. The enzyme activity was deterrnined at 300C by following the rate ofNADH oxidation at 340 nm in a Hitachi 124 spectrophotometer, as in the case ofPEP carboxylase (7).   2. Assay ef the enzyme in the directien of decarboxylation-The standardreaction mixture contained 5 ptmoles of oxaloacetate, 2 Itmoles of ATP, 80 ptraolesof MgS04, O.2 ptmole of DTT and 70 ptmoles of Tris--maleate buffer, pl{ 7.0, in atotal volume of 1.0 ml. The mixture was incubated at 300C for 10 min and thenit was cooled in an ice bath to stop the enzyme reaction. The enzyme activitywas assayed by determination of inorganic phosphate which was liberated fromPEP, one of the reaction products, by the addition of O.1 ml of 50/. HgC12 to thereaction mixture (8). Inorganic phosphate was determined by the method ofYoda (9).   One unit of the enzyme was defined as the amount of producing 1 pamole ofproduct per min under the assay conditions.Protein Determination   Protein concentration was determined by the method of Lowry et al. (10)with crystalline bovine serum albumin as a standard.PARTIAL PURIFICATION AND PROPERTIES OF PHOSPHOENOLPYRUVATE 143            CARBOXYKINASE OF ESCLEIERICHJA COLI W                 RESULTS AND DISCUSSIONPurification of the Enzyme    All operations were performed at O-4eC. The crude cell-free extract was dilutedwith O.l M Tris-HCI buffer, pH 7.5, containing 1 mM EDTA and O.4 mM DTT toa final concentration of 10 mg protein per ml and the solution was treated withO.25 volume of IO/. protamine sulfate solution. The suspension was stirred for lemin and centrifuged at 15,OOOxg for 10 min. To the supernatant solution wasadded solid ammonium sulfate and the fraction precipitating between 55 to 750/.saturation was dissolved in a minimal volume of O. 1 M Tris-HCI buffer, pH 7.5,containing 1 mM EDTA and O.4 rnM DTT. The enzyme solution was dialyzedovernight against 170 volumes of O.Ol M potassium phosphate buffer, pH 6.0, con-taining O.1 mM DTT. Suspension of calcium phosphate gel washed twice with O.05M potassium phosphate buffer, pH 6:O, containing O.l rmvi DTT (20 mg dry weight/ml) was added to the dialyzate in a proportion of 2 mg dry gel to 1 mg protein withgentle stirring. The mixture was further stirred for 5 min and then centrigufed topack the gel. After the gel was washed once with O.05 M potassium phosphatebuffer, pH 6.0, containing e.1 mM DTT, the combined supernatant was broughtto 80% saturation with solid ammonium sulfate. The precipitated fraction wasdissolved in e.05 M Tris-HCI buffer, pH 7.7, containing O.05 M KCI, 1 mM EDTAand O.l mM DTT. The enzyme solution was dialyzed overnight against 180 volumesof the same buffer and applied to a DEAE-Sepltadex column (1.5 Å~ 40 cm) previouslyequilibrated with the buffer. The enzyme was eluted with a linear gradient of KCIconcentration from O.05 to O.5 M in O.05 M Tris-HCI buffer, pH 7.7, containing 1mM EDTA and O.4 mM DTT and fractions of 4ml were collected (Fig. 1). Thefractions from 32 to 39 were pooled and solid ammonium sulfate was added to thesolution to a level of 800/. saturation. The precipitate was centrifuged and dis-solved in a minimal volume of O.1 M Tris-HCI buffer, pH 7.7, containing 1 mMEDTA and O.4 mM DTT. The enzyme solution was applied to a Sephadex G-leOcolumn (1.1Å~60 cm) previously equilibrated with the same buffer. The enzymewas eluted with the buffer and O.6-ml fractions were collected. Fractions with the5g,ii'  xxxxXXLf xlNx   x.t'1"xKr}st,x t''x,fxxx##  x,l8"x '"gxNxxxfrkxxxE.ofuo'k  .5oO.6O.4  --s  E  vO.2 n  -o  5Fig. 1.    ve.-.t-ww,bmu..-. 6 o Oo     IO 20 50 40 50 60        Fraction Number(4-ml fraction)Elution pattern of PEP carboxykinase from DEAE-Sephadex columR.-g- Enzyme activity;-Å~-, absorbance at 280 nm; , KCIconcentratlon.144 H. TERAOI<A, K. IZUI and H. KATSUKIspecific activity higher than 8 units per ing of protein were pooled and used for theexperiments described below.   Table 1 shows one oftke typical results ofthe purification. PEP carboxykinasewas purified 85-fold over the crude cell-free extract of E. coli W.Table 1. Purification ofPEP carboxykiRaseStep Protem (mg)Activitya) (units) Specitic actlvity(unitslmg)Yield(O/e)Purification  (-fold)1. Crude extract2. Protamine sulfate3. (NH4)2S04  (55-75 % saturation)4, Calcitim phosphate gei5. DEAE-Sephadex  ckromatography6. Sepahdex G-100  chromatography30581826520gi18,4323319276??l78.0O.IlO.i7e.43l:9.31eo9986gg24gl8l85a) Assayed in a direction of C02-fixation reaction.Kinetie Properties   1• C02-fixation reaction-pH optimum of CO,-fixation reaction was 7.0 inO•07 M Tris-maleate buffer. The activity was abeut one-half its maximum valueat pH 5.5 and about one-fifth at pH 8.0. As shown in Fig. 2A, Mn2" was essentialfor the enzyme activity and the Km value was O.6 mM. When Mg2' was usedinstead of Mn2', the enzyme activity attainable at its infinite concentration wasonly IO-20% of that attainable with Mn2". Figure 2 (B-D) shows effects of vary-ing concentrations of substrates on tke reactioR velocity. Irhe curve showing tliereaction velocity vs. PEP concentration was hyperbolic and the Km valtie for PEPwas O.6 mM (Fig. 2B). As for ADP (Fig. 2C) and KHCO, (Ng. 2D), curves showiiagthe reaction velocity vs. the substrateconcentrations were hyperbolic and Km valuesor ADP and KHCO, were O.4 mM and 40 mM, respectively.   It is interesting to consider the relation between the physiological function ofthe enzyme and its kinetic properties. in vivo systern of E. coli, the enzyme reac-tion is presumed not to operate in a direction of C02-fixation, because a mutantdevoid of PEP carboxylase [EC 4.1.1.31] which catalyzes the reaction for replenish-ing oxaloacetate from PEP has been reported to be unable to grow on glucose,glycerol or pyruvate as sole carbon source in spite of the possession of PEP car-boxykinase (ll). This fact suggests that a certain unknown mechanism is involvedin the control of PEP carboxykinase reaction in the cells (4). When consideredtlie result obtained above, it seems possible that the Iow aMnity of PEP carboxyki-nase for KHC03 contributes significantly to the control of the enzyme reaction inthe C02-fixation direction in yivo. In the E. eoli B enzyme, Km valt}es for ADPand PEP were reported to be O.23 mM and 14 mM, respectively (5).   2. Decarboxylatien reactien-In the experiments for tlte decarboxylation re-action, Mg2+ was used as bivalent cation, because Mn2+ accelerated the non-enzy-matical decarboxylation of oxaloacetate. The pH profile of the reaction velocitywas considerably broad in a range of 6.0 to 8.0, showing a pH optimam of 7.0.Figure 3A shows oxaloacetate-saturation curve of the reaction velocity, indicatingPART!ALPURIFICATION AND PROPERTIES OF PHOSPHOENOLPYRUVATE itl5    CAR.BOXYKINASE OF ESCHERICHIA COLI W30    B20      os'AsO,4O.2 ee5Q          t---+05         IO  ll(MnClal{mM-')     5(Mn Cl2) (mM)IO20'giPtsi,t..s02O.leooo 8 -t}Åí P]( ,4,l!I2 IOl 2[PEP)345(mM) 6 7ac     ocO,2so.i6po    5 IOll {ADp] (mM-i)5040 o 3'gzi's'Dso,iQ05O O.I. 02 11 [l<HC03] {mM-i), Fig. 2.'l 2 5 4 5 OO IO 20 so 40 50•  [ADP] <,nM) CKHC03} (rriM} Effects of MnCl2, PEP, ADP and KHC03 on the C02-fixation reaction of PEP carboxykinase. The reaction mixtures in A, B, C aRd D contained 2, 3, 2 and 5 ng protein ofthe enzyme, respectively. Other experimental conditions were as described in "Materials and Methods" except for the use of ligand conceRtrations as iRdicated in the abscissa. Reaction velocity was expressed as nmoles of C02-fixed per min. Insets represent 'double reciprocal plots of reaction velocity and ligand concentration.Km value for oxaloacetate te be O.6 mM. Km value'for ATP was estimated to beabout-e.2 mM, and an inhibition was observed at higher concentratiOns of ATP                                '                    'than•l mM (Fig. '3B).' ' • '• ..'Me}ecular and Regniatory Properties    The molecular wight of the enzyme was estirnated to be 64,OOO froin the ex-periments using Sephadex G-IOO gel filtration (l2). This value was identical withthat of E. eoli B (5), which was estimated by the method of Sephadex .G-l50 gelfiltration and of sucrose density gradient centrifugation. No repoft on the subunitstructure of the enzyme has appeared.146 H. TERAOKA, K. IZUI and H. KATSUKIIO      5'iiA to      5'i-B              oo        O l 2 3 4 5 O l 2 3 4                   (Oxaloacetate) (mM) (ATP) (mM)    Fig. 3. Effects of oxaloacetate and ATP on the decarboxylation reaction of PEP          carboxykinase. Tke reaction mixture contained 10 ptg protein of the enzyme.          ExperimeRtal conditions were as described in "Materials and Methods"          except for the use of substrate concentrations as indicated in the abscissa.          Reaction ve}ocity was expressed as nmoles of PEP formed per min.    The enzyme activity was inhibited by SH-blocking reagents-p mercuribenzoateand N-ethylmaleimide, as in the case of the yeast enzyme (13). Dilution of theenzyme preparation with Tris-HCI buffer, pH 7.5, resulted jn a drastic decreasein the enzyme activity. If DTT at a concentration of O.1 mM or mgre was presentin the buffer for dilution, a complete protection against the inactivation due todilution was observed. These results indicate that the enzyme from E. coli Wcontains essential SH group(s) for the activity.    Since PEP carboxykinase is one of the key enzymes in gluconeogenesis, it seems1ikely that the enzyme is presumed to be a regulatory one. Studies on the controlof the enzyme level by several groups of investigators including us (2-4) indicatethat the enzyme level in E. coli is high in gluconeogenesis and low in glycolysis.As for the control ef the enzyme actiyity, there is no evidence supporting that PEPcarboxykinase of microbial origin has allosteric effector(s), except for the E. coliB enzyrne which is regulated with NADH in an allosteric manner (5). So far asexamined in a direction of decarboxylation reaction, the E. coli W enzyme was notsignificantly inhibited by NADH. This result is in confiict with the data of theE. coli B enzyme whose activity was assayed by determining oxaloacetate by thecoloration with Azoene fast violet B. However, NADH (O,5 mM-l mM) directlyinhibited the coloration of oxaloacetate, whereas NAD" did not inhibit in a rangeof concentration of oxaloacetate from O.05 to O.5 mM (not shown). Therefore,apparent inhibition of the enzyme by NADH observed by Wright and Sanwalmight be due to the inhibition of color development by NADH.   The activity of E. coli W enzyme was unaffectedby some metabolites, such asglucose 6-phosphate, fructose 1,6-bisphosphate, succinate, aspartate, citrate, 5'-AMP, and 3',5'--cyclic AMP. •                           REFERENCES1) H. A. Lardy, D. O. Foster, E. Shrago and P. D. Ray (1964) in Advaiices in Enzyme Regtttation,   ed by G. Weber, Vol. 2, p. 39-47.2) A. W. Hsie and H. V. Rickenberg (1966) Biochem. Biophys. Res. Commun. 25, 676-683.3) E. Shrago and A. L. Shug (1969) Arch. Biochem. Biophys. 130, 393-398.4)5)6)7)8)9)lO)11)i2)l3)PART{AL PURIFICATION AND PROPERTIES OF PHOSPHOENOLPYRUVATE 147          CARBOXYKINASE OF ESCIEIERICHIA COLI W H. Teraoka, T. Nishikido, K. Izui and K. Katsuki (l970) J. Biochern. 67, 567-575. J. A. Wright and B. D. Sanwal (1969) J. BioL Chem. 244, 1838-1845. T. Nishikido, K. Izui, A. Iwatani, H. Katsuki and S. Tanaka (1968) J. Biochetn. 63, 532-541. T. Yoshinaga, K. Izui aRd H. Katsuki (1970) J. Biochem. 68, 747-750. K. Lohrnann and O. Meyerhof (l934) Biochem. Z. 273, 60-72. A. Yoda (1955) Nippon Kagalcu Zasshi (in Japanese) 8e, 488-490. O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall (1951) J. BioL Chem. 193, 265- 275. J. M. Ashworth aRd H. L. Kornberg (1966) Proc. Roy. Soc. B l65, 179-188. P. G. Squire (1964) Arelt. Biochem. Biophys. 107, 471--478. J. J. B. Cannata and A. O. M. Stoppani (1963) J. Biol. Chem. 238, 1208-1212.

Partial Purification and Properties of Phosphoenolpyruvate Carboxykinase of Escherichia Coli W

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Partial Purification and Properties of Phosphoenolpyruvate Carboxykinase of Escherichia Coli W

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