Download Fructokinase (Fraction III)of Pea Seeds

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
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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.
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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
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mM
m
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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
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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.
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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
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