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The pH-dependent substrate specificity of UDP-glucose anthocyandin 3-rhamnosylglucoside, 5-O-glucosyltransferase in petals of Silene dioica: the formation of anthocyanidin 3,5-diglucosides
Department of Population and Evolutionary Biology, University of Utrecht, Padualaan 8, Utrecht, The Netherlands
The pH-Dependent Substrate Specificity of UDP-glucose: anthocyandin 3-rhamnosylglucoside, 5-0-glucosyltransferase in Petals of Silene dioica: The Formation of Anthocyanidin 3,5-diglucosides JOHN KAMSTEEG, JAN VAN BREDERODE
and
GERRIT VAN NIGTEVECHT
With 2 figures Received July 2, 1979 . Accepted August 8, 1979
Summary Depending upon the pH, the enzyme UDP-glucose: anthocyanidin 3-rhamnosylglucoside, s-O-glucosyltransferase catalyzes the formation of either anthocyanidin 3-rhamnosylglucoside-s-glucosides or 3,s-diglucosides. Maximal formation of anthocyanidin 3-rhamnosylglucoside-s-glucosides takes place at pH 7.4; of anthocyanidin 3,s-diglucosides at pH 6.5. The reaction rate of the S-O-glucosylation of cyanidin 3-rhamnosyl-glucoside at pH 7.4 was stimulated 1.6 fold by 1 mM CaCI 2 • Contrary, the glucosylation of cyanidin 3-glucoside at pH 6.5 was not stimulated by divalent metal ions. The «true Km» at pH 7.4 for cyanidin 3-rhamnosylglucoside is 3.6 mM. At pH 6.5 a Km value of 23.4 mM for cyanidin 3-glucoside was found. The affinity for UDP-glucose was not influenced by the pH; both at pH 6.5 and 7.4 the «true Km» for UDP-glucose was O.6mM. Key words: Silene dioica, Caryophyllaceae, anthocyanins, glucosyltransferase, pH, metal ions, substrate specificity.
Introduction
The enzyme UDP-glucose: anthocyanidin 3-rhamnosylglucoside, 5-0-glucosyltransferase isolated from petals of Silene dioica plarrts of the genotype M/M N/N, catalyzes the formation of cyanidin-, or pelargonidin 3-rhamnosylglucoside from cyanidin-, respectively pelargonidin 3-rhamnosylglucoside and UDP-glucose. The formation of this enzyme is controlled by gene M; in petals of mlm plants no glucosyltransferase activity is found. In the assay system as described by KAMSTEEG et al. (1978 a), the radioactivity from UDP-(U-14C)glucose incorporated into cyanidin 3,5-diglucoside did not exceed twice the background. Therefore it was concluded that the enzyme was unable to use cyanidin 3-glucoside as a substrate. However, this anthocyanin can be synthesized in petals of M/M n/n, S. dioica plants. Z. Pflanzenphysiol. Bd. 96. S. 87-93. 1980.
88
JOHN KAMSTEEG, JAN VAN BREDERODE
and
GERRIT VAN NIGTEVECHT
In petals of these plants besides cyanidin 3-g1ucoside,cyanidin 3,5-dig1ucoside is present. When the protein preparation isolated from this genotype was tested it also failed to synthesize anthocyanidin 3,5-dig1ucosides. So either another enzyme than the 5-0-glucosyltransferase controlled by gene M catalyzes this reaction, or the enzyme controlled by gene M in addition catalyzes the 5-0-glucosylation of cyanidin 3-glucoside under other conditions. In this paper these two possibilities are further investigated. Material and Methods Preparation of substrates The preparation of substrates was carried out as described before
(KAMSTEEG
et aI., 1978).
Enzyme preparation All operations were carried out at 4 0c. Five grams petals were homogenized in an allglass Potter-Elvehjem homogenizer with 5 ml of 20 mM p-mercaptoethanol, 5 % PVP, 1 0/0 Triton X-I00, 50 mM potassium-sodium phosphate buffer (pH 7.4) and centrifuged for 15 min at 38,000 X g. To remove phenolic compounds, other lower molecular weight substances, and PVP, the supernatant fraction was subsequently passed through a Polyclar AT column (1 by 30 em) and a Sephadex G-50 column (2.5 by 35 em), which had been equilibrated before use with a 4 mM p-mercaptoethanol, 0.02 % Triton X-I00, 10 mM potassium-sodium phosphate buffer (pH 7.4). The protein fraction was concentrated in an Amicon on-line concentrator (CEC 1) with an UM 10 filter (Amicon, Massachusetts). This fraction was used in the determination of the enzyme properties. Assay of enzyme activity The standard reaction mixture consisted of 25,u1 enzyme in 4 mM p-mercaptoethanol, 0.02 Ofo Triton X-I00, 10 mM potassium-sodium phosphate buffer (pH 7.4), 10,u1 13.8 mM' cyanidin 3-glucoside or cyanidin 3-rhamnosylglucoside in 0.1 mM hydrochloric acid, 16 mmol glycylglycine buffer of pH 6.5 or 7.4, 10,u1 9.1 mM UDP-(U- 14 C) glucose (S.A. 5 Ci/mol) and CaCl 2 at a final concentration of 1 mM. After incubation of the reaction mixture for 15 minutes at 30°C, the reaction was stopped by the addition of 50,u1 10 Ofo trichloroacetic acid in methanol. Subsequently, the reaction mixture was transferred to Whatman III, together with the carriers cyanidin 3,5-diglucoside and/or cyanidin 3rhamnosylglucoside-5-glucoside, and chromatographed two-dimensionally in n-butanol : acetic acid: water (4 : 1 : 5, v/v/v, upper phase) and 1 % hydrochloric acid in water, respectively. The portion of the chromatogram bearing the carrier was cut out, placed in a scintillation vial with 20 ml toluene liquifluor, and counted in a Packard Tricarb Spectrometer. The tests were run in duplicate. For determination of the zero-control trichloroacetic acid was added to the reaction mixture before incubation.
Results and Discussion
To get more insight in the biosynthesis of anthocyanidin 3,5-dig1ucosides in petals of Silene dioica plants, the sensitivity of the 5-0-glucosylating system for the formation of cyanidin 3,5-diglucoside was enhanced by using UDP-(U- 14 C)gIucose
z. Pflanzenphysiol. Bd. 96. S. 87-93. 1980.
89
Formation of anthocyanidin 3,5-diglucosides
with a S-times higher specific activity compared to KAMSTEEG et al. (1978 a). With this improved system we found sufficient incorporation of radioactivity into cyanidin 3,S-diglucoside for further investigation of its synthesis. These studies show that maximal cyanidin 3,S-diglucoside formation took place at approximately one pH value lower (pH 6.5, Fig. 1 a; solid line) than the optimum found for the formation of cyanidin 3-rhamnosylglucoside-S-glucoside (pH 7.4, Fig. 1 b; solid line). The S-O-glucosylation of cyanidin 3-rhamnosylglucoside is stimulated by divalent metal ions (Fig. 2). These ions are without any effect on the formation of cyanidin 3,S-diglucoside. EDTA (1 mM, pH 7.5) inhibited the formation of cyanidin 3-rhamnosylglucosideS-glucoside at pH 7.4, but had no effect on the formation of cyanidin 3,5diglucoside at pH 6.5. The divalent metal ions Zn2+ and Hg2+ inhibited the 5-0glucosylation of both substrates at pH 6.5 and 7.4.
<;>
<;'I
o
~ ~10
~5 ~
~
!!l c
!!l c
:>
:>
o
u
t
o
8
u
t
,,
..,,,
6
4
0 ----~-o-----.Q
'q, ,,
.
3
,,
--'-1:]0,0.,
'11(1..
,
4
,, ,
,, ,
2
A _pH
_pH
(e-e)
Fig. 1: pH-dependence (cyanidin 3-glucoside and cyanidin 3-rhamnosylglucoside (0-0) and pH denaturation curves [cyanidin 3-glucoside ( . - . ) and cyanidin 3rhamnosylglucoside (0-0)] of 5-0-glucosylation. The reaction mixture contained in a total volume of 60.aI, 125 nmol potassium-sodium phosphate, 50 nmol p-mercaptoethanol, 0.01 Ofo Triton X-I00, 28 nmol cyanidin 3-glucoside, respectively cyanidin 3-rhamnosylglucoside, 18 nmol UDP-(U- 14C)glucose (S.A. 5 Ci/moi), 16 mmol glycyiglycine buffer of the indicated pH-values, and CaCi 2 in a final concentration of 1 mM. The reaction mixture was incubated for 15 minutes at 30°C. Incubation mixtures were prepared in parallel (UDP-glucose omitted) to determine the pH. Z. Pflanzenphysiol. Bd. 96. S. 87-93. 1980.
90
JOHN KAMSTEEG, JAN VAN BREDERODE
, 8
and
GERRIT VAN NIGTEVECHT
,
7
6
-.- -..... -.-._-- -4 ._. - ' ..--.-._._
C")
._._.
-.- .... -.-. _._0
_._-_.- .•
o x 5 c
E "til
+oJ
C
::J
83
--
- --- ---.,
'.
--- --6- - - - - - - :"-,-:--6
2
'.
°O~----------~5----------~1~O----------~15~--------~20~
Me 2 + mM Fig. 2: Effect of divalent metal ions on UDP-glucose: anthocyanidin 3-rhamnosylglucoside, 5-0-glucosyltransferase activity. The reaction mixture contained in a total volume of 50,u1, 125 nmol potassium-sodium phosphate buffer (pH 7.4), 50 nmol Jl-mercaptoethanol, 18 nmol UDP-(U-14C)glucose (S.A. 5 Cilmol), om Ofo Triton X-l00, 28 nmol cyanidin 3-rhamnosylglucoside and the divalent metal ions in the final concentrations as indicated: CaCI2 (e-e), MnCl 2 (0-0), CoCl 2 ( . - . ) , MgCl 2 (.6-.6) and ZnCl 2 (A-A).
The conversion of the respective anthocyanin substrates exhibited Michaelis and Menten kinetics (LINEWEAVER and BURK, 1934). At the different substrate concentrations used, the apparent Km values for the anthocyanin substrate depended upon the concentration of the second substrate UDP-glucose. The «true Km» values were determined according to FLORINI and VESTLING (1957). From Table 1 it follows that only the affinity and specificity for the anthocyanin acceptor varied with the pH, and not the affinity for the glucose donor, UDP-glucose. The formation of cyanidin 3-rhamnosylglucoside-5-glucoside proceeds 5-10 fold faster than the cyanidin 3,5-diglucoside formation. This difference was also found with pelargonidinglycosides. Z. P/lanzenphysiol. Bd. 96. S. 87-93. 1980.
91
Formation of anthocyanidin 3,S-diglucosides
Table 1: True Michaelis Menten constants and V max of UDP-glucose: anthocyanidin 3rhamnosylglucoside, S-O-glucosyltransferase for various substrates at two pH-values. pH
Km and Vmax of substrates
6.5
UDP-glucose Cyanidin 3-glucoside V max
7.4
UDP-glucose Cyanidin 3-glucoside Vmax
0.67 mM 23.4 mM 0.4 nmol
UDP-glucose Cy 3-rhamnosylglucoside V max
0.59 mM 32 mM 4.3 nmol
UDP-glucose Cy 3-rhamnosylglucoside V max
0.5 3.6 33.7
mM mM nmol
"-) The various kinetic parameters could not be determined, due to the very low conversion of cyanidin 3-glucoside into cyanidin 3,S-diglucoside at this pH. Vmax in nmol. min- 1 .mg protein-1 •
These differences in conditions for the S-O-glucosylation of anthocyanidin 3-rhamnosylglucoside and anthocyanidin 3-glucoside, respectively, point to the presence of two different enzymes. However, the following findings strongly suggest that both activities depend on one enzyme: (i) the pH-denaturation curves for both S-O-glucosylation reactions coincide (Fig. 1 a, b; dashed lines). (ii) both reactions are controlled by gene M; protein extracts of petals of m/m plants are neither able to catalyze the formation of cyanidin 3,S-diglucoside nor the formation of cyanidin 3-rhamnosylglucoside-S-glucoside. Both petal extracts of M/M n/n plants, which in vivo contain a mixture of cyanidin 3-glucoside and cyanidin 3,S-diglucoside, and of M/M N/N plants in which cyanidin 3-rhamnosylglucoside-S-glucoside is found, the same pH-dependent substrate specificity is exhibited. (iii) after gelfiltration over a calibrated Sephadex G-1S0 column, the formation of both cyanidin 3,S-diglucoside and cyanidin 3-rhamnosylglucoside-S-glucoside took place in the same protein fraction corresponding with a molecular weight of approximately 55,000 daltons. The stimulation by divalent metal ions of the S-O-glucosylation of cyanidin 3-rhamnosylglucoside possesses a sharp optimum at 1 mM (Fig. 2). This stimulation correlates with the ionic radius of the ions tested (Table 2). A shift of one pH unit probably leads to such a change in the tertiairy structure of the enzyme that the metal ion binding site is no longer available or has lost its significance. The binding site for the anthocyanidin-glycoside acceptor has changed as well. At pH 6.5, in addition to cyanidin 3-rhamnosylglucoside, cyanidin 3-glucoside can now be used as substrate. At this pH value the affinity of the enzyme for both substrates is about equal, but 5-10 fold lower than that of cyanidin 3-rhamnosylglucoside at pH 7.4. The affinity of the binding site for UDP-glucose has not changed. Changes in substrate specificity depending on pH have been shown before in plants. SALEH et al. (1978) found that the hydroxylation pattern of the aromatic ring of hydroxycinnamoyl-CoA substrates has a strong influence upon the pH at which the flavanone synthetase, present in cell suspension cultures of Haplopappus gracilis Z. P/lanzenphysiol. Bd. 96. S. 87-93. 1980.
92
JOHN KAMSTEEG, JAN VAN BREDERODE and GERRIT VAN NIGTEVECHT
Table 2: Relationship between ion radius and stimulation of the reaction rate of the enzyme UDP-glucose: anthocyanidin 3-rhamnosylglucoside, 5-0-glucosyltransferase at pH 7.4 with cyanidin 3-rhamnosylglucoside as substrate. divalent metal ion
ion radius':-)
Ca 2+ Mn 2+ Co2+ Mg2+ Zn 2+ Hg2+ none (EDTA)
0.99 0.80 0.72 0.66 0.74 1.10
A
0/0
total acti vi ty
183 176 140 132 70 28 100
The reaction mixture contained in a total volume of 50,ul, 125 nmol potassium-sodium phosphate buffer (pH 7.4), 16 mmol glycylglycine buffer pH 7.4, 50 nmol ,B-mercaptoethanol, 18 nmol UDP-(U- 14 C)glucose (S.A. 5 Ci/mol), 0.Q1 Ofo Triton X-100, 28 nmol cyanidin 3rhamnosylglucoside and the divalent metal ions as indicated in the final concentration of 1 mM. The reaction mixture was incubated for 15 minutes at 30°C. The total activity is expressed as percentage of the assay with 1 mM EDTA. ,:-) WEAST (1974/1975). or parsley, expresses its maximal actIVIty. In Silene alba the 2" -O-glucosylation of isoorientin, a C-glucosylflavone with two hydroxyl groups in the B-ring, exhibits its maximal activity at pH 7.5. At pH 8.5 this glucosylation is hardly detectable, but at this pH isovitexin, a C-glucosylflavone with one hydroxyl group in the B-ring, is used as substrate (VAN BREDERODE et al., 1979). The 2" -O-glucosylation of both isovitexin and isoorientin is controlled by one single gene Fg. The physiological significance of these pH-dependent differences in substrate specificity is unknown. They might be involved in a mechanism for the separation of analogous biochemical pathways. The reason why always about equal amounts of cyanidin 3,S-diglucoside and cyanidin 3-glucoside are found in petals of M/M n/n plants could not be clarified. Addition of cyanidin 3,S-diglucoside in concentrations ranging from 1-10 mM to the assay had no influence upon the reaction rate. We therefore suggest that the 1 : 1 ratio is a result of a steady state mechanism. This investigation was supported by a grant from the Research Pool of the University of Utrecht.
References BREDERODE, J. VAN, J. CHOPIN, J. KAMSTEEG, G. VAN NIGTEVECHT, and R. HEINSBROEK: The pH-dependent substrate specificity of UDP-glucose: Isovitexin 2" -O-glucosyltransferase in Silene alba. Phytochemistry 18, 655-656 (1979). FLORINI, J. R. and C. S. VESTLING: Graphical determination of the dissociation constants for two substrate enzyme systems. Biochim. Biophys. Acta 25, 575-578 (1957). Z. Pjlanzenphysiol. Bd. 96. S. 87-93. 1980.
Formation of anthocyanidin 3,5-diglucosides
93
KAMSTEEG, J., J. VAN BREDERODE, and G. VAN NIGTEVECHT: Identification and properties of UDP-glucose: Cyanidin 3-0-glucosyltransferase isolated from petals of the Red Campion (Silene dioica). Biochem. Gen. 16, 1045-1058 (1978). - - - Identification, properties and genetic control of UDP-glucose: Cyanidin 3-rhamnosyl (1 -+ 6) glucoside, 5-0-glucosyltransferase isolated from petals of the Red Campion (Silene dioica). Biochem. Gen. 16, 1059-1071 (1978 a). LINEWEAVER, H. and D. BURK: The determination of enzyme dissociation constants. J. Am. Chern. Soc. 56, 658-666 (1934). SALEH, N. A. M., H. FRITSCH, F. KREUZALER, and H. GRISEBACH: Flavanone synthase from cell suspension cultures of Haplopappus gracilis and comparison with the synthase from parsley. Phytochemistry 17, 183-186 (1978). WEAST, R. c.: Handbook of chemistry and physics, 55th ed., CRC Press, Cleveland (19741 1975). Dr. G. VAN NIGTEVECHT, Department of Population and Evolutionary Biology, Padualaan 8, Utrecht, The Netherlands.
Z. Pjlanzenphysiol. Bd. 96. S. 87-93. 1980.
The pH-Dependent Substrate Specificity of UDP-glucose: anthocyandin 3-rhamnosylglucoside, 5-0-glucosyltransferase in Petals of Silene dioica: The Formation of Anthocyanidin 3,5-diglucosides JOHN KAMSTEEG, JAN VAN BREDERODE
and
GERRIT VAN NIGTEVECHT
With 2 figures Received July 2, 1979 . Accepted August 8, 1979
Summary Depending upon the pH, the enzyme UDP-glucose: anthocyanidin 3-rhamnosylglucoside, s-O-glucosyltransferase catalyzes the formation of either anthocyanidin 3-rhamnosylglucoside-s-glucosides or 3,s-diglucosides. Maximal formation of anthocyanidin 3-rhamnosylglucoside-s-glucosides takes place at pH 7.4; of anthocyanidin 3,s-diglucosides at pH 6.5. The reaction rate of the S-O-glucosylation of cyanidin 3-rhamnosyl-glucoside at pH 7.4 was stimulated 1.6 fold by 1 mM CaCI 2 • Contrary, the glucosylation of cyanidin 3-glucoside at pH 6.5 was not stimulated by divalent metal ions. The «true Km» at pH 7.4 for cyanidin 3-rhamnosylglucoside is 3.6 mM. At pH 6.5 a Km value of 23.4 mM for cyanidin 3-glucoside was found. The affinity for UDP-glucose was not influenced by the pH; both at pH 6.5 and 7.4 the «true Km» for UDP-glucose was O.6mM. Key words: Silene dioica, Caryophyllaceae, anthocyanins, glucosyltransferase, pH, metal ions, substrate specificity.
Introduction
The enzyme UDP-glucose: anthocyanidin 3-rhamnosylglucoside, 5-0-glucosyltransferase isolated from petals of Silene dioica plarrts of the genotype M/M N/N, catalyzes the formation of cyanidin-, or pelargonidin 3-rhamnosylglucoside from cyanidin-, respectively pelargonidin 3-rhamnosylglucoside and UDP-glucose. The formation of this enzyme is controlled by gene M; in petals of mlm plants no glucosyltransferase activity is found. In the assay system as described by KAMSTEEG et al. (1978 a), the radioactivity from UDP-(U-14C)glucose incorporated into cyanidin 3,5-diglucoside did not exceed twice the background. Therefore it was concluded that the enzyme was unable to use cyanidin 3-glucoside as a substrate. However, this anthocyanin can be synthesized in petals of M/M n/n, S. dioica plants. Z. Pflanzenphysiol. Bd. 96. S. 87-93. 1980.
88
JOHN KAMSTEEG, JAN VAN BREDERODE
and
GERRIT VAN NIGTEVECHT
In petals of these plants besides cyanidin 3-g1ucoside,cyanidin 3,5-dig1ucoside is present. When the protein preparation isolated from this genotype was tested it also failed to synthesize anthocyanidin 3,5-dig1ucosides. So either another enzyme than the 5-0-glucosyltransferase controlled by gene M catalyzes this reaction, or the enzyme controlled by gene M in addition catalyzes the 5-0-glucosylation of cyanidin 3-glucoside under other conditions. In this paper these two possibilities are further investigated. Material and Methods Preparation of substrates The preparation of substrates was carried out as described before
(KAMSTEEG
et aI., 1978).
Enzyme preparation All operations were carried out at 4 0c. Five grams petals were homogenized in an allglass Potter-Elvehjem homogenizer with 5 ml of 20 mM p-mercaptoethanol, 5 % PVP, 1 0/0 Triton X-I00, 50 mM potassium-sodium phosphate buffer (pH 7.4) and centrifuged for 15 min at 38,000 X g. To remove phenolic compounds, other lower molecular weight substances, and PVP, the supernatant fraction was subsequently passed through a Polyclar AT column (1 by 30 em) and a Sephadex G-50 column (2.5 by 35 em), which had been equilibrated before use with a 4 mM p-mercaptoethanol, 0.02 % Triton X-I00, 10 mM potassium-sodium phosphate buffer (pH 7.4). The protein fraction was concentrated in an Amicon on-line concentrator (CEC 1) with an UM 10 filter (Amicon, Massachusetts). This fraction was used in the determination of the enzyme properties. Assay of enzyme activity The standard reaction mixture consisted of 25,u1 enzyme in 4 mM p-mercaptoethanol, 0.02 Ofo Triton X-I00, 10 mM potassium-sodium phosphate buffer (pH 7.4), 10,u1 13.8 mM' cyanidin 3-glucoside or cyanidin 3-rhamnosylglucoside in 0.1 mM hydrochloric acid, 16 mmol glycylglycine buffer of pH 6.5 or 7.4, 10,u1 9.1 mM UDP-(U- 14 C) glucose (S.A. 5 Ci/mol) and CaCl 2 at a final concentration of 1 mM. After incubation of the reaction mixture for 15 minutes at 30°C, the reaction was stopped by the addition of 50,u1 10 Ofo trichloroacetic acid in methanol. Subsequently, the reaction mixture was transferred to Whatman III, together with the carriers cyanidin 3,5-diglucoside and/or cyanidin 3rhamnosylglucoside-5-glucoside, and chromatographed two-dimensionally in n-butanol : acetic acid: water (4 : 1 : 5, v/v/v, upper phase) and 1 % hydrochloric acid in water, respectively. The portion of the chromatogram bearing the carrier was cut out, placed in a scintillation vial with 20 ml toluene liquifluor, and counted in a Packard Tricarb Spectrometer. The tests were run in duplicate. For determination of the zero-control trichloroacetic acid was added to the reaction mixture before incubation.
Results and Discussion
To get more insight in the biosynthesis of anthocyanidin 3,5-dig1ucosides in petals of Silene dioica plants, the sensitivity of the 5-0-glucosylating system for the formation of cyanidin 3,5-diglucoside was enhanced by using UDP-(U- 14 C)gIucose
z. Pflanzenphysiol. Bd. 96. S. 87-93. 1980.
89
Formation of anthocyanidin 3,5-diglucosides
with a S-times higher specific activity compared to KAMSTEEG et al. (1978 a). With this improved system we found sufficient incorporation of radioactivity into cyanidin 3,S-diglucoside for further investigation of its synthesis. These studies show that maximal cyanidin 3,S-diglucoside formation took place at approximately one pH value lower (pH 6.5, Fig. 1 a; solid line) than the optimum found for the formation of cyanidin 3-rhamnosylglucoside-S-glucoside (pH 7.4, Fig. 1 b; solid line). The S-O-glucosylation of cyanidin 3-rhamnosylglucoside is stimulated by divalent metal ions (Fig. 2). These ions are without any effect on the formation of cyanidin 3,S-diglucoside. EDTA (1 mM, pH 7.5) inhibited the formation of cyanidin 3-rhamnosylglucosideS-glucoside at pH 7.4, but had no effect on the formation of cyanidin 3,5diglucoside at pH 6.5. The divalent metal ions Zn2+ and Hg2+ inhibited the 5-0glucosylation of both substrates at pH 6.5 and 7.4.
<;>
<;'I
o
~ ~10
~5 ~
~
!!l c
!!l c
:>
:>
o
u
t
o
8
u
t
,,
..,,,
6
4
0 ----~-o-----.Q
'q, ,,
.
3
,,
--'-1:]0,0.,
'11(1..
,
4
,, ,
,, ,
2
A _pH
_pH
(e-e)
Fig. 1: pH-dependence (cyanidin 3-glucoside and cyanidin 3-rhamnosylglucoside (0-0) and pH denaturation curves [cyanidin 3-glucoside ( . - . ) and cyanidin 3rhamnosylglucoside (0-0)] of 5-0-glucosylation. The reaction mixture contained in a total volume of 60.aI, 125 nmol potassium-sodium phosphate, 50 nmol p-mercaptoethanol, 0.01 Ofo Triton X-I00, 28 nmol cyanidin 3-glucoside, respectively cyanidin 3-rhamnosylglucoside, 18 nmol UDP-(U- 14C)glucose (S.A. 5 Ci/moi), 16 mmol glycyiglycine buffer of the indicated pH-values, and CaCi 2 in a final concentration of 1 mM. The reaction mixture was incubated for 15 minutes at 30°C. Incubation mixtures were prepared in parallel (UDP-glucose omitted) to determine the pH. Z. Pflanzenphysiol. Bd. 96. S. 87-93. 1980.
90
JOHN KAMSTEEG, JAN VAN BREDERODE
, 8
and
GERRIT VAN NIGTEVECHT
,
7
6
-.- -..... -.-._-- -4 ._. - ' ..--.-._._
C")
._._.
-.- .... -.-. _._0
_._-_.- .•
o x 5 c
E "til
+oJ
C
::J
83
--
- --- ---.,
'.
--- --6- - - - - - - :"-,-:--6
2
'.
°O~----------~5----------~1~O----------~15~--------~20~
Me 2 + mM Fig. 2: Effect of divalent metal ions on UDP-glucose: anthocyanidin 3-rhamnosylglucoside, 5-0-glucosyltransferase activity. The reaction mixture contained in a total volume of 50,u1, 125 nmol potassium-sodium phosphate buffer (pH 7.4), 50 nmol Jl-mercaptoethanol, 18 nmol UDP-(U-14C)glucose (S.A. 5 Cilmol), om Ofo Triton X-l00, 28 nmol cyanidin 3-rhamnosylglucoside and the divalent metal ions in the final concentrations as indicated: CaCI2 (e-e), MnCl 2 (0-0), CoCl 2 ( . - . ) , MgCl 2 (.6-.6) and ZnCl 2 (A-A).
The conversion of the respective anthocyanin substrates exhibited Michaelis and Menten kinetics (LINEWEAVER and BURK, 1934). At the different substrate concentrations used, the apparent Km values for the anthocyanin substrate depended upon the concentration of the second substrate UDP-glucose. The «true Km» values were determined according to FLORINI and VESTLING (1957). From Table 1 it follows that only the affinity and specificity for the anthocyanin acceptor varied with the pH, and not the affinity for the glucose donor, UDP-glucose. The formation of cyanidin 3-rhamnosylglucoside-5-glucoside proceeds 5-10 fold faster than the cyanidin 3,5-diglucoside formation. This difference was also found with pelargonidinglycosides. Z. P/lanzenphysiol. Bd. 96. S. 87-93. 1980.
91
Formation of anthocyanidin 3,S-diglucosides
Table 1: True Michaelis Menten constants and V max of UDP-glucose: anthocyanidin 3rhamnosylglucoside, S-O-glucosyltransferase for various substrates at two pH-values. pH
Km and Vmax of substrates
6.5
UDP-glucose Cyanidin 3-glucoside V max
7.4
UDP-glucose Cyanidin 3-glucoside Vmax
0.67 mM 23.4 mM 0.4 nmol
UDP-glucose Cy 3-rhamnosylglucoside V max
0.59 mM 32 mM 4.3 nmol
UDP-glucose Cy 3-rhamnosylglucoside V max
0.5 3.6 33.7
mM mM nmol
"-) The various kinetic parameters could not be determined, due to the very low conversion of cyanidin 3-glucoside into cyanidin 3,S-diglucoside at this pH. Vmax in nmol. min- 1 .mg protein-1 •
These differences in conditions for the S-O-glucosylation of anthocyanidin 3-rhamnosylglucoside and anthocyanidin 3-glucoside, respectively, point to the presence of two different enzymes. However, the following findings strongly suggest that both activities depend on one enzyme: (i) the pH-denaturation curves for both S-O-glucosylation reactions coincide (Fig. 1 a, b; dashed lines). (ii) both reactions are controlled by gene M; protein extracts of petals of m/m plants are neither able to catalyze the formation of cyanidin 3,S-diglucoside nor the formation of cyanidin 3-rhamnosylglucoside-S-glucoside. Both petal extracts of M/M n/n plants, which in vivo contain a mixture of cyanidin 3-glucoside and cyanidin 3,S-diglucoside, and of M/M N/N plants in which cyanidin 3-rhamnosylglucoside-S-glucoside is found, the same pH-dependent substrate specificity is exhibited. (iii) after gelfiltration over a calibrated Sephadex G-1S0 column, the formation of both cyanidin 3,S-diglucoside and cyanidin 3-rhamnosylglucoside-S-glucoside took place in the same protein fraction corresponding with a molecular weight of approximately 55,000 daltons. The stimulation by divalent metal ions of the S-O-glucosylation of cyanidin 3-rhamnosylglucoside possesses a sharp optimum at 1 mM (Fig. 2). This stimulation correlates with the ionic radius of the ions tested (Table 2). A shift of one pH unit probably leads to such a change in the tertiairy structure of the enzyme that the metal ion binding site is no longer available or has lost its significance. The binding site for the anthocyanidin-glycoside acceptor has changed as well. At pH 6.5, in addition to cyanidin 3-rhamnosylglucoside, cyanidin 3-glucoside can now be used as substrate. At this pH value the affinity of the enzyme for both substrates is about equal, but 5-10 fold lower than that of cyanidin 3-rhamnosylglucoside at pH 7.4. The affinity of the binding site for UDP-glucose has not changed. Changes in substrate specificity depending on pH have been shown before in plants. SALEH et al. (1978) found that the hydroxylation pattern of the aromatic ring of hydroxycinnamoyl-CoA substrates has a strong influence upon the pH at which the flavanone synthetase, present in cell suspension cultures of Haplopappus gracilis Z. P/lanzenphysiol. Bd. 96. S. 87-93. 1980.
92
JOHN KAMSTEEG, JAN VAN BREDERODE and GERRIT VAN NIGTEVECHT
Table 2: Relationship between ion radius and stimulation of the reaction rate of the enzyme UDP-glucose: anthocyanidin 3-rhamnosylglucoside, 5-0-glucosyltransferase at pH 7.4 with cyanidin 3-rhamnosylglucoside as substrate. divalent metal ion
ion radius':-)
Ca 2+ Mn 2+ Co2+ Mg2+ Zn 2+ Hg2+ none (EDTA)
0.99 0.80 0.72 0.66 0.74 1.10
A
0/0
total acti vi ty
183 176 140 132 70 28 100
The reaction mixture contained in a total volume of 50,ul, 125 nmol potassium-sodium phosphate buffer (pH 7.4), 16 mmol glycylglycine buffer pH 7.4, 50 nmol ,B-mercaptoethanol, 18 nmol UDP-(U- 14 C)glucose (S.A. 5 Ci/mol), 0.Q1 Ofo Triton X-100, 28 nmol cyanidin 3rhamnosylglucoside and the divalent metal ions as indicated in the final concentration of 1 mM. The reaction mixture was incubated for 15 minutes at 30°C. The total activity is expressed as percentage of the assay with 1 mM EDTA. ,:-) WEAST (1974/1975). or parsley, expresses its maximal actIVIty. In Silene alba the 2" -O-glucosylation of isoorientin, a C-glucosylflavone with two hydroxyl groups in the B-ring, exhibits its maximal activity at pH 7.5. At pH 8.5 this glucosylation is hardly detectable, but at this pH isovitexin, a C-glucosylflavone with one hydroxyl group in the B-ring, is used as substrate (VAN BREDERODE et al., 1979). The 2" -O-glucosylation of both isovitexin and isoorientin is controlled by one single gene Fg. The physiological significance of these pH-dependent differences in substrate specificity is unknown. They might be involved in a mechanism for the separation of analogous biochemical pathways. The reason why always about equal amounts of cyanidin 3,S-diglucoside and cyanidin 3-glucoside are found in petals of M/M n/n plants could not be clarified. Addition of cyanidin 3,S-diglucoside in concentrations ranging from 1-10 mM to the assay had no influence upon the reaction rate. We therefore suggest that the 1 : 1 ratio is a result of a steady state mechanism. This investigation was supported by a grant from the Research Pool of the University of Utrecht.
References BREDERODE, J. VAN, J. CHOPIN, J. KAMSTEEG, G. VAN NIGTEVECHT, and R. HEINSBROEK: The pH-dependent substrate specificity of UDP-glucose: Isovitexin 2" -O-glucosyltransferase in Silene alba. Phytochemistry 18, 655-656 (1979). FLORINI, J. R. and C. S. VESTLING: Graphical determination of the dissociation constants for two substrate enzyme systems. Biochim. Biophys. Acta 25, 575-578 (1957). Z. Pjlanzenphysiol. Bd. 96. S. 87-93. 1980.
Formation of anthocyanidin 3,5-diglucosides
93
KAMSTEEG, J., J. VAN BREDERODE, and G. VAN NIGTEVECHT: Identification and properties of UDP-glucose: Cyanidin 3-0-glucosyltransferase isolated from petals of the Red Campion (Silene dioica). Biochem. Gen. 16, 1045-1058 (1978). - - - Identification, properties and genetic control of UDP-glucose: Cyanidin 3-rhamnosyl (1 -+ 6) glucoside, 5-0-glucosyltransferase isolated from petals of the Red Campion (Silene dioica). Biochem. Gen. 16, 1059-1071 (1978 a). LINEWEAVER, H. and D. BURK: The determination of enzyme dissociation constants. J. Am. Chern. Soc. 56, 658-666 (1934). SALEH, N. A. M., H. FRITSCH, F. KREUZALER, and H. GRISEBACH: Flavanone synthase from cell suspension cultures of Haplopappus gracilis and comparison with the synthase from parsley. Phytochemistry 17, 183-186 (1978). WEAST, R. c.: Handbook of chemistry and physics, 55th ed., CRC Press, Cleveland (19741 1975). Dr. G. VAN NIGTEVECHT, Department of Population and Evolutionary Biology, Padualaan 8, Utrecht, The Netherlands.
Z. Pjlanzenphysiol. Bd. 96. S. 87-93. 1980.