Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
NADH:ubiquinone oxidoreductase (H+-translocating) wikipedia , lookup
Metalloprotein wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Citric acid cycle wikipedia , lookup
Adenosine triphosphate wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Glyceroneogenesis wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Plant Physiol. (1978) 62, 291-294 Fructokinase (Fraction III) of Pea Seeds' Received for publication February 6, 1978 and in revised form April 25, 1978 LES COPELAND, DOROTHY D. HARRISON, AND JOHN F. TURNER Department of Agricultural Chemistry, University of Sydney, N.S. W 2006, Australia ABSTRACT A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sadvum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millilar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent Km values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent Km for Mg adenosine 5'triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg2e was essential for activity but the enzyme was inhibited by excess Mg2e or ATP. Mg adenosine 5'-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K+, Rb+, and NH4' were the most effective. The possible role of fructokinase (fraction III) is discussed. properties of a different enzyme, fructokinase (fraction III), which was purified from fraction III. This enzyme will be referred to as fructokinase III. Fructokinase III was specific for fructose as substrate and had little activity with glucose. The enzyme was inhibited by fructose (in concentrations greater than 0.25 mM) and by ATP, Mg2+, MgATP, and MgADP. Glucose-6-P and fructose6-P were only slightly inhibitory. MATERIALS AND METHODS Materials. Mature pea seeds (Pisum sativum L. var. Progress No. 9) were obtained from F. Cooper Ltd., Wellington, New Zealand. Glucose-6-P dehydrogenase, P-glucose isomerase, pyruvate kinase, lactate dehydrogenase, NADP, NADH, ATP, ADP, P-enolpyruvate, Tris, D-glucose, D-fructose, D-galactose, D-mannose, L-sorbose, D-mannoheptulose, D-tagatose, D-xylose, D-glucosamine, 2-deoxy-D-glucose, sucrose, and DEAE-cellulose were obtained from Sigma Chemical Co. or Boehringer Mannheim GmbH. Purification of Fructokinase III. Pea seeds were extracted and The properties of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from yeast and mammalian tissues have been the hexose kinases fractionated on a DEAE-cellulose column as studied extensively (2, 15). Bakers' yeast contains two native described previously (20). The fractions containing the first peak hexokinases, one of which (form P-II) may be activated by low of fructose-phosphorylating activity (fraction III), but none of the concentrations of several metabolites (2, 10). Four isozymes of fraction IV fructokinase, were pooled and concentrated to apmammalian hexokinase have been identified and the proportions proximately 10 ml by ultrafiltration in a Diaflo apparatus (XM-50 of these vary in different tissues (2, 15, 16). A feature of three of membrane, 300 kPa/m2 nitrogen pressure). The concentrate was the forms of mammalian hexokinase is strong inhibition by glu- dialyzed against 10 mm Tris-HCl buffer (pH 8) and placed on a cose-6-P and this phenomenon is believed to be important in the DEAE-cellulose column (1.5 x 20 cm) previously equilibrated regulation of hexokinase activity (2). The fourth enzyme form is with the same buffer. Fructokinase III was eluted with a gradient a glucokinase (ATP:D-glucose 6-phosphotransferase, EC 2.7.1.2) obtained by introducing 200 ml of 10 mm Tris-HCl (pH 8) which was first found in liver (7, 18). A ketohexokinase (ATP:D- containing 0.30 M KCI and 2 mim EDTA, into 200 ml of 10 mM fructose I-phosphotransferase, EC 2.7.1.3), which phosphorylates Tris-HCl (pH 8). Fractions of 5 ml were collected. Fructokinase fructose to give fructose-l-P, is also present in liver (3, 8, 14). This III appeared as a single peak of activity which was eluted with 0.20 M KCI. The fractions containing fructokinase III were pooled enzyme is sometimes referred to as a "fructokinase." Saltman (19) demonstrated hexokinase activity in extracts from and preparations of this type, which contained approximately 0.4 several plant tissues and later workers have also reported the mg of protein/ml, were used to obtain the results presented in this presence of the enzyme in plants (1, 4-6, 9, 13). Medina and Sols paper. Protein was determined in all fractions (except the crude (12) noted the presence of a fructokinase (ATP:D-fructose 6-phos- extract) by measuring the A at 280 nm. An absorption of 1.0 in a photransferase, EC 2.7.1.4) in immature pea seeds. Fructokinase 10-mm light path was taken to be equivalent to 1 mg of protein/ml. activity has been observed also in nectaries (6) and the conducting The enzyme preparation could be stored frozen at - 18 C for at least 2 months without loss in activity but if stored at 4 C only bundles of sugar beet leaf petioles (1 1). The situation in plants with respect to hexose kinases may be 50%o of activity remained after 24 hr. Freezing and thawing after comparable in some ways to that in mammalian tissues. In an elution from the first DEAE-cellulose column resulted in 30%o loss earlier publication it was shown that mature pea seeds contain of fructose-phosphorylating activity. Assay of Fructokinase Activity. Enzyme activity was assayed by several hexose kinases (20). Four fractions, designated I, II, III, and IV in order of elution from a DEAE-cellulose column, were coupling the production of glucose-6-P (derived from fructose-6obtained from (NH4)2SO4 fractions of pea seed extracts. Fraction P) with the reduction of NADP in the presence of excess glucoseI contained a glucokinase which had Km values of 0.07 mm and 30 6-P dehydrogenase. Reaction mixtures for the standard assay mm for glucose and fructose, respectively (20). The fraction IV contained, in a total volume of 3 ml, 75 ,umol of Tris-HCI buffer enzyme, fructokinase IV, was also characterized (21). This enzyme, (pH 8), 0.6 ,umol of fructose, 3 timol of ATP, 4.5 ,umol of MgC12, a specific fructokinase with low activity toward glucose and man- 1 t,mol of NADP, 3 ,ug of P-glucose isomerase, 0.6 ,ug of glucosenose, had a requirement for K+ ions. Here, we describe the 6-P dehydrogenase, and a volume of the fructokinase III preparation containing 30 to 50 ,ug of protein. When glucose was used ' This investigation was supported by the University of Sydney Research as substrate P-glucose isomerase was omitted. Reaction mixtures Grant. were maintained at 30 C and the increase in A at 340 nm was Downloaded from on June 16, 2017 - Published by www.plantphysiol.org 291 of Plant Biologists. All rights reserved. Copyright © 1978 American Society 292 COPELAND, HARRISON, AND TURNER TABLE I Purification of Fructokinase III Ether-extracted pea powder (40 g) was treated as described in the text. Reaction mixtures were of the composition described for the standard assay. Stage Protein Fructokinase Activity Specific Activity mg mu mU/mg protein 2640 96001 Content 30-50% (NH4)2 50 1st DEAEcellulose Ultrafiltration 2nd DEAEcellulose 3.9 61 2200 36 42 2000 48 12 1125 94 Plant Physiol. Vol. 62, 1978 approximately equal concentrations. The enzyme was inhibited by an excess of either MgCl2 or ATP. Figure 3 shows the effect on fructokinase III activity of increasing MgCl2 concentration. When the concentration of MgATP was varied (in the presence ofa fixed excess of 0.5 mm MgCl2), the activity of fructokinase III increased until the MgATP concentration was 1.0 mM (Fig. 4). With higher MgATP concentrations the enzyme showed substrate inhibition. The apparent Km of fructokinase III for MgATP was 0.06 mm under the standard assay conditions. Inhibition by excess MgATP was also observed in assays in imidazole-HCI buffer at pH 6.6 (Fig. 4). Effect of pH. Fructokinase III showed a broad pH optimum when assayed under standard conditions in a series of imidazoleHCl and Tris-HCI buffers. The optimum pH was 8.0 and 90% or 'Includes fructokinases III and IV followed. One mU2 of fructokinase activity is defined as 1 nmol of glucose-6-P produced/min. Fructokinase activity was directly proportional to the amount of pea seed enzyme used in the assay. The enzyme preparation was free of 6-P-gluconate dehydrogenase and no phosphatase activity with p-nitrophenyl-P at pH 8 was detected. When the phosphorylation of sugars other than fructose and glucose was examined, the production of ADP was coupled with the oxidation of NADH using pyruvate kinase and lactate dehydrogenase. This method of assay was also used for investigation of the effects of glucose-6-P and fructose-6-P on fructokinase III activity. Reaction mixtures contained in a total volume of 1 ml, 25 ,umol of Tris-HCI buffer (pH 8), 20 ,umol of KCl, 1 ,umol of ATP, 1.5 ,umol of MgCl2, 1 pmol of P-enolpyruvate, 0.14 ,umol of NADH, 8 ,ug of pyruvate kinase, 16 ,ug of lactate dehydrogenase, a volume of the fructokinase III preparation containing 30 to 50 ,ug protein, and the substrate sugar. No phosphatase activity with P-enolpyruvate or ATP was detected. 80 im 0p E 40 E 260 1 2 3 4 5 3 4 5 < 20 0 0 1 2 Fructose concn (mM) FIG. 1. Effect of fructose concentration on pea seed fructokinas InI activity. Reaction mixtures at pH 8 were of the composition described for the standard assay with the concentration of fructose varied as shown. RESULTS Reaction mixtures at pH 6.6 contained 75 Ismol of imidazole-HCI buffer. 0. pH 8; 0: pH 6.6. When the DEAE-cellulose column fractions were assayed under the standard conditions, fraction III contained approximately three times as much fructose-phosphorylating activity as fraction IV. After elution from the second DEAE-cellulose column, the 10 F fructokinase III preparations had specific activities of approxiA A mately 90 mU/mg of protein. The fructose-phosphorylating activ.c8 ities at different stages of a typical purification are shown in Table I. Effect of Hexose Concentration. The activity of fructokinase III E increased until the fructose concentration reached 0.20 mM but with concentrations higher than 0.25 mm the enzyme showed strong substrate inhibition (Fig. 1). With a fructose concentration of 1.5 mm, the activity was only 50% of that obtained with 0.20 <2 mm fructose. The apparent Km value of fructokinase III for fructose was 0.06 mm at pH 8. In contrast to fructokinase IV (21), 0 substrate inhibition with fructose was also observed when the 12 4 6 8 10 0 2 enzyme was assayed in imidaole-HCl buffer at pH 6.6 (Fig. 1). Glucose concn (mM) The optimum concentration of fructose at pH 6.6 was 0.25 mM. FIG. 2. Phosphorylation of glucose by pea seed fructokinase III. ReFructokinase III also phosphorylated glucose but the maximum action mixtures were of the composition described for the standard assay rate was only 9% of that obtained with fructose (Fig. 2). No with fructose replaced by glucose in the concentrations shown. the and with was observed inhibition apparent substrate glucose Km was 0.14 mM. A comparison of the relative activities of TABLE II fructokinase III with fructose and glucose is given in Table II. DKinetic Constants of Fructokinase III Mannose, 2-deoxy-D-glucose, and D-glucosamine were phosphoReaction mixtures were of the composition rylated to a small extent and there was slight 'activity with the described for the standard assay with the varied. hexose ketoses D-tagatose and D-xylose (Table III). Effect of Concentration of ATP and M{+. Fructokinase III km V/km V Hexose showed no activity in the absence of Mg2+ and phosphorylation mM mU/mg protein of fructose was maximal when ATP and MgCl2 were present in 1633 0.060 0- 98 D-Fructose 8.6 D-Glucose V: maximum from velocity. Downloaded on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1978 American Society of Plant Biologists. All rights reserved. 2Abbreviations: mU: milliunit; 0.14 61 TABLE III Substrate Specificity of Fructokinase III Reaction mixtures were of the composition described for the standard assay with pyruvate kinase and lactate dehydrogenase as Hexose concentrations coupling enzymes . were 5 mM unless otherwise shown. Phosphorylating Hexose Activity mU/mg protein 129 42 9.1 5.1 D-Fructose (0.2 mM) D-Fructose D-Glucose D-Mannose D-Tagatose 1.1 0.9 0.0 0.0 0.0 0.0 6.9 7.4 D-Xylose 0-Mannoheptulose L-Sorbose D-Galactose Sucrose 2-Deoxy-D-glucose D-Glucosamine 80 so 60 c2 0 E E40 E >1 u 293 PEA SEED FRUCTOKINASE III Plant Physiol. Vol. 62, 1978 20 obtained with 1.5 mm MgADP. Increasing the concentration of fructose increased the extent of inhibition by MgADP, e.g. when the fructose concentration was 1 mM, 1.5 mM MgADP inhibited fructokinase III activity by approximately 70%. In contrast, increase in concentration of the other substrate, MgATP, decreased the inhibition given by MgADP (Fig. 5). The sigmoid curve in the presence of 5 mM MgATP suggests that MgADP may function as a cooperative inhibitor. A sigmoid curve was also observed in some assays when the MgATP concentration was 2 mM. In separate experiments it was found that UDP was less inhibitory than ADP: under conditions of the standard assay, 1.5 mM MgUDP inhibited pea seed fructokinase III by 10%. Effect of Metabolites. Both glucose-6-P (final concentration 6 mm) and fructose-6-P (6 mM) inhibited fructokinase III in standard assay reaction mixtures by 25%. The inhibition given by glucose6-P and fructose-6-P was less than 5% when the concentration of either of these metabolites was 1.5 mm. Effect of Monovalent Cations. Fructokinase III as prepared contained approximately 200 mM KC1. In experiments where the effect of auded monovalent cations was studied the enzyme preparation was dialyzed against 10 mm Tris-HCI buffer (pH 8) for 90 min and the coupling enzymes dialyzed in the same way. Reaction mixtures prepared from the dialyzed fructokinase III and coupling enzymes contained 0.2 mM K4, 3.0 mM Na+, and 0.7 mM NH44. Table IV shows the effect of the addition of chlorides of monovalent cations on fmctokinase III activity. With no addition the activity was 47 mU/mg of protein and the addition of K+, Rb+, or NH4+ (final concentration 60 mM) stimulated fructokinase III activity by 53 to 60%. Na+, Li+, and Cs+ were much less effective stimulators of fructokinase III and at a final concentration of 60 100 0 4 3 2 Mg Cl2 concn (mMI 80 c 0 FIG. 3. Effect of Mg2+ concentration on pea seed fructokinase III activity. Reaction mixtures were of the composition described for the standard assay with the concentration of MgCl2 varied as shown. 80 60 ._ z c st 40 [ 20 0 0 _ 60 FIG. 5. Effect of MgADP and MgATP concentration on pea seed fructokinase III activity. Reaction mixtures were of the composition described for the standard assay at different concentrations of ATP and MgC12 (plus 0.5 mm excess MgCl2) and with MgADP added as shown. 0: 1.0 mM MgATP; 0: 2.0 mm MgATP; R 5.0 mm MgATP. L0' co, Er 4 3 2 1 Mg ADP concn (mM) .0 40 E >1 < 20 TABLE IV Effect of Monovalent Cations on Fructokinase III Activity 0 2 6 4 MgATP concn 8 10 (mM) FIG. 4. Effect of MgATP concentration on pea seed fructokinase III activity. Reaction mixtures at pH 8 were of the composition described for the standard assay with ATP and MgC12 (plus 0.5 mM excess MgC12) varied as shown. Reaction mixtures at pH 6.6 contained 75 pmol of imidazole-HCI buffer. 0: pH 8; 0: pH 6.6. Reaction mixtures were of the composition described for the standard assay. The fructokinase III preparation and the coupling enzymes were dialyzed as described in the text. Chlorides of monovalent cations were added to give the final concentrations shown. With no addition the fructokinase activity was 47 mU/mg protein. Addition Fructokinase III mU/mg 60 mM of the maximum activity was obtained between pH 7.4 and 72 K+ 72 Rb+ pH 8.4. 75 NH*,+ 49 Na+ Effect of ADP. Fructokinase III was inhibited by ADP. Under 52 Li+ (0.2 the standard ATP, and fructose, I 46 Cs+ IIIonactivity fructokinase from June 16,was 2017 - Published by www.plantphysiol.org 1.5 mm Mg24), 50% inhibition ofDownloaded more mM m Copyright © 1978 American Society of Plant Biologists. All rights reserved. Activity protein 120 mM 71 69 70 58 60 55 294 COPELAND, HARRISON, AND TURNER mm showed little effect; when the final concentration was increased to 120 mm there was a 17 to 28% stimulation of enzyme activity. Plant Physiol. Vol. 62, 1978 inhibition became cooperative with increase in MgATP concentration. The concentration of ADP in developing pea seeds may reach 1 mm (17), a potentially inhibitory level. ADP is a product of the ADP-glucose-starch synthase reaction and regulation of fructokinase III by ADP could possibly be of metabolic signifiDISCUSSION cance. The inhibitory effect of concentrations of fructose higher This investigation has shown that pea seeds contain a second than 0.25 mm raises the possibility of inhibition of fructokinase specific fructokinase, fructokinase III, which is different from the III by levels of fructose which could be expected in peas; in fructokinase IV described previously (21). The maximum rate of developing pea seeds the concentration of fructose ranges from phosphorylation of fructose was 11 times the maximum rate 1.3 to 21 mM (24). obtained with glucose and the value of the expression V/Km with Acknowledgments-The authors wish to thank S. G. Letton and C. J. Vockler for technical fructose as substrate was 27 times the figure with glucose. Glucose was an even poorer substrate for fructokinase IV: with this enzyme assistance. the maximum rate with fructose was 38 times that given by glucose LITERATURE CITED and the value of V/Km with fructose was 263 times the value obtained with glucose (21). The apparent Km values for fructose 1. AKAZAWA T, T MINAMIKAWA, T MURATA 1964 Enzymic mechanism of starch synthesis in ripening rice grains. Plant Physiol 39: 371-378 of fructokinase III (0.06 mM) and fructokinase IV (0.057 mM) were 2. COLOWICK SP 1973 The hexokinases. Enzymes 9: 1-48 very similar. GT, S OCHOA, MW SLEIN, CF CORI 1951 The metabolism of fructose in liver. Isolation Pea seed fructokinase III was inhibited by the substrate fructose 3. CORI of fructose-l-phosphate and inorganic pyrophosphate. Biochim Biophys Acta 7: 304-317 in concentrations above 0.25 mm at the standard assay pH (8.0) 4. Cox EL, DB DICKINSON 1971 Hexokinases from maize endosperm. Phytochemistry 10: 1771-1775 and also at pH 6.6. On the other hand, fructokinase IV was subject EL, DB DICIUNSON 1973 Hexokinases from maize endosperm and scutellum. Plant to fructose inhibition at the optimum pH (8.2) but not at pH 6.6 5. CoxPhysiol 960-966 (21). Fructokinase III was inhibited at pH 8.0 by excess of the 6. DE FEKETE51:MAR, H ZIEGLER, R WOLF 1967 Enzyme des Kohlenhydratstoffwechsels in other substrate, MgATP: in these experiments an excess of 0.5 mm Nektarien. Planta 75: 125-138 DL, C SHARMA, S WEINHOUSE 1962 Studies on glucose phosphorylation in rat liver. Mg2e was maintained over the total ATP concentration to reduce 7. DIPIETRo Biochemistry 1: 455-462 the proportion of free ATP. Fructokinase III was also inhibited 8. HERS 1952 La fructokinase du foie. Biochim Biophys Acta 8: 416-423 by Mg2 in excess of the ATP concentration and by ATP in excess 9. JACOBHG J-L, J D'AUzAC 1967 Sur 1'existence conjointe d'une hexokinase et d'une fructokinase of the Mg2+ concentration. With fructokinase IV there was little au sein du latex d'Hevea brasiliensis. CR Acad Sci 265: 260-263 inhibition by MgATP at pH 8.2 (21). Fructokinase III had consid- 10. Kosow DP, IA RosE 1971 Activators of yeast hexokinase. J Biol Chem 246: 2618-2625 AL, SV SOKOLOVA, MV TURKINA 1970 Hexokinase in conducting tissue of sugarerable activity in the absence of added monovalent cations but 11. KURSANOV beet and its possible connection with transport of sugars through cell membranes. J Exp Bot K+, which could be partially replaced by Na+, stimulated the 21: 30-39 enzyme by more than 50%o. In contrast, pea seed fructokinase IV 12. MEDINA A, A SOLs 1956 A specific fructokinase in peas. Biochim Biophys Acta 19: 378-379 was almost inactive without added monovalent cations in the 13. MEUNIER JC, J Buc, J RICARD 1971 Isolation, purification and characterization of wheat germ hexokinases. FEBS Lett 14: 25-28 reaction mixture and showed a specific requirement for K+ ions 14. PARKS RE, E BEN-GERSHOM, HA LARDY 1957 Liver fructokinase. J Biol Chem 227: 231-242 (21). 15. PURICH DL, HJ FROMM, FB RUDOLPH 1973 The hexokinases: kinetic, physical and regulatory The total fructose-phosphorylating capacity in pea seed extracts properties. Adv Enzymol 39: 249-326 is greater than the total glucose-phosphorylating capacity (20). 16. RosE IA, ZB ROSE 1969 Glycolysis: regulation and mechanism of the enzymes. In M Florkin, EH Stotz, eds, Comprehensive Biochemistry, Vol 17. Elsevier, Amsterdam, pp 93-161 During the development of the pea seed there is a substantial KS, DH TURNER 1957 Physiology of pea fruits. V. Phosphate compounds in the conversion of sucrose to starch (22) and the initial step in this 17. ROWAN developing seed. Aust J Biol Sci 10: 414-425 process is believed to be catalyzed by sucrose synthase yielding 18. SALAS J, M SALAS, E VINUELA, A SOLS 1965 Glucokinase of rabbit liver. Purification and properties. J Biol Chem 240: 1014-1018 fructose and UDP-glucose (23). The fructose formed in this reachigher plants. J Biol Chem 200: 145-154 tion must be converted to fructose-6-P for entry into glycolysis, 19. SALTMAN P 1953 HexokinaseDDin HARRISON 1977 Glucokinase of pea seeds. Biochim Biophys IF, QJ CHENSEE, polysaccharide synthesis, etc. This points to a requirement for 20. TURNER Acta 480: 367-375 considerable fructose-phosphorylating activity. There may be a 21. TURNER JF, DD HARRISON, L COPELAND 1977 Fructokinase (fraction IV) of pea seeds. Plant Physiol 60: 666-669 smaller demand for glucose phosphorylation during most of the 22. TURNER IF, DH TURNER 1957 Physiology of pea fruits. III. Changes in starch and starch period of growth of the pea seed. phosphorylase in the developing seed. Aust J Biol Sci 10: 302-309 There seems to be potential for regulation of pea seed fructo- 23. TURNER JF, DH TURNER 1975 The regulation of carbohydrate metabolism. Annu Rev Plant kinase III since the enzyme was inhibited by Mge, free ATP and Physiol 26: 159-186 MgATP, and was stimulated by monovalent cations. Pea seed 24. TURNER JF, DH TURNER, JB LEE 1957 Physiology of pea fruits. IV. Changes in sugars in the developing seed. Aust J Biol Sci 10: 407-413 fructokinase III was sensitive to inhibition by MgADP and this Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1978 American Society of Plant Biologists. All rights reserved.