Planta (1985)165:59-67
P l a n t a 9 Springer-Verlag1985
Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena* A. Yokota, S. Kitaoka, K. Miura and A. Wadano Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 591, Japan
Abstract. The nonenzymatic reaction of glyoxylate and H202 was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H202 was secondorder, with a rate constant of 2.271 tool-1 s-1 at pH 8.0 and 25 ~ C. The rate constant increased by 4.4 times in the presence of Zn 2 + and doubled at 35 ~ C. We propose a mechanism for the reaction between glyoxylate and HzO 2 . From a comparison of the rates of H202 decomposition by catalase and the reaction with glyoxylate, we conclude that H202 produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO2 originates from glycine, but not from glyoxylate, in C 3 plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO 2 . In the simulation of the glycolate pathway of E u g l e n a , the rate constant was high enough to ensure the decarboxylation of glyoxylate by H202 to produce photorespiratory C O 2 during the glycolate metabolism of this organism. Key words: C 3 plant - E u g l e n a - Glycolate pathway (simulation) - Glyoxylate. * This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982)
Abbreviations. Chl = chlorophyll; GGT = glutamate: glyoxylate aminotransferase (EC2.6.1.4); Hepes=4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; SGT = serine: glyoxylate aminotransferase (EC 2.6.1.45)
Introduction
Glycolate, the substrate of photorespiration in C 3 plants, is produced through the action of ribulose1,5-bisphosphate carboxylase/oxygenase and phosphoglycolate phosphatase in chloroplasts, transferred to peroxisomes, and oxidized by glycolate oxidase (EC 1.1.3.1; for a review, see Tolbert 1980). The product of the oxidation, glyoxylate, may take part in one of the following three reactions; re-reduction to glycolate by the glycolateglyoxylate shuttle (Tolbert et al. 1970); decarboxylation by H20 2 in chloroplasts (Zelitch 1972) andor peroxisomes (Halliwell and Butt 1974; Grodzinski 1978); and transamination to glycine in peroxisomes (Rehfeld and Tolbert 1972). The existence of the glycolate-glyoxylate shuttle is obscured in C 3 plants because of the large difference of the pH optimum of chloroplast NADPH: glyoxylate reductase from the stromal pH under illumination and the low concentration of glyoxylate in situ (Schnarrenberger and Fock 1976). The oxidative decarboxylation of glyoxylate by H20 2 has received special attention in the metabolic elucidation of the evolution of photorespiratory CO 2 (Halliwell 1981; Zelitch 1971), but the occurrence of glutamate: glyoxylate aminotransferase (GGT) in peroxisomes and the rapid oxidation of glycine in mitochondria have tended to deny the involvement of non-enzymic glyoxylate decarboxylation in photorespiration (Chollett 1977; Ogren 1984). How glyoxylate escapes from oxidative degradation by H20 2 in peroxisomes has nor been elucidated (Tolbert 1980) because of lack of precise information on the reactivity of glyoxylate and H202, which has not been studied except for the
60
A. Yokota et al. : Reactivity of glyoxylate and HzO 2 in glycolate pathway
glycolate (A)
glycerate (F)
~
glycolate (A)
v2 hydroxypyruvate {El
O2 - ~ 8 H 2 0
+
1/2 02
v H202
"~"-~ glyoxylate (B)" v7
~
CO2 (G) + formate
NH2-d~176 v3
g l y /
Vcin~ ,cl
serine (D)
e (D)
~'~4
Perox isome
glycine (C)
CO2 (H) + NH3
itochondrion report of Hatcher and Holden (1925) who used extremely high concentrations of glyoxylate and H20 2 , and non-physiological pH's. Euglena gracilis, a photosynthetic protozoon, decarboxylates the carboxyl carbon of glycolate with no conversion to glycine during glycolate metabolism under highly photorespiratory conditions (Yokota and Kitaoka 1982). Glyoxylate formed by glycolate dehydrogenase (EC 1.1.99.14) in mitochondria may be decarboxylated in chloroplasts by H20 2 produced by a Mn 2 +-dependent N A D P H oxidase of chloroplasts (Yokota et al. 1983). We have measured the rate constant of the reaction between glyoxylate and H202, and simulated the glycolate-pathway kinetics of C 3 plants and Euglena using the rate constant we determined and published information. Our objective was to re-evaluate and possibly confirm the importance of the glyoxylate decarboxylation by H202 in the glycolate pathway of C 3 plants and Euglena. Materials and methods Materials. Glyoxylate was purchased from Sigma Chemical Co. (St. Louis, Mo., USA) and U 2 0 z from Nakarai Chemicals Co. (Kyoto, Japan). The concentration of glyoxylate was determined by measuring the oxidation of NADH with lactate dehydrogenase fromyeast and that of H20 z by light absorbancy
Fig. 1. Metabolic pathway of glycolate in C3-plant cells. Stoichiometries of the reactions are not taken into account in this figure. Kinetic parameters used in the simulation are as follows in the order: name of the metabolic step, maximum rate of the enzymatic reaction in gmol rag- 1 Chl h - 1, Km value for the substrate in mM: rate of the glycolate influx, vl, 60 (Zelitch 1971); glycolate oxidase, v2, 100, 0.38 for glycolate (Zelitch 1955; 1971); GGT, v3, 30, 1.2 for glyoxylate (Noguchi and Hayashi 1981; Yamazaki and Tolbert 1970; Zelitch 1971); alanine:glyoxylate aminotransferase, v3, 95, 1.2 for glyoxylate (Noguchi and Hayashi 1981); conversion of glycine to serine, v4, 100, 1.0 for glycine (Gardestr6m et al. 1980; Moore et al. 1977); SGT, vs, 20, 2.72 for serine, 0.15 for glyoxylate (Noguchi and Hayashi 1980); hydroxypyruvate reductase, v6, 80, 0.12 for hydroxypyruvate (Tolbert 1971; Tolbert et al. 1970). The estimations of the maximum reaction rates assumed 1.5 mg Chl = 1 g fresh weight =0.5 d i n / = 3 mg nitrogen = 20 mg protein, according to Zelitch (1971)
(McEwen 1971) and by the method of Hosoya and Morrison 1967) using peroxidase. Assay of reaction of glyoxylate and H202 . The reaction of glyoxylate and H202 was followed by measuring the initial decrease of light absorbance of H20 z (within 2 s). The extinction coefficient of H/O 2 at this wavelength is 61 1 mol-1 cm- t according to McEwen (197J). Since glyoxylate also absorbs light at 240 nm and the extinction coefficient of glyoxylate at this wavelength was 141mol - I cm -x, the apparent decrease of the absorbance at 240 nm was divided by 1.23 to give the actual decrease of the absorbance of H20 z in the reaction mixture. The decrease of the absorbance at 240 nm was followed with a recording spectrophotometer (model 200-100; Hitachi, Tokyo, Japan). Separation and determination of reactants and reaction products. The 14C02evolved from [l-l*C]glyoxylate was determined by the method of Yokota et al. (1978). After the reaction, the mixture was passed through a column (0.8 cm diameter, 2 cm long) of Dowex 50 (H+-type: BioRad Laboratories, Tokyo, Japan) to obtain the free-acid forms of the reactants and reaction products. Formic and glyoxylic acids were separated from each other by high-performance liquid chromatography (HPLC) with a Shodex C8t I column (Showa Denko, Japan) as reported in Yokota et al. (1983), and the recovery of formate was the same as that of glyoxylate.
Kinetic model Figure 1 shows the glycolate pathway with the intermediates, reaction rates and kinetic parameters which have been reported. v 1 is the rate of the influx of glycolate from chloroplasts into the glycolate pathway, v2 to v6 represent enzymatic reaction rates, v: is the reaction velocity between glyoxylate and HzO 2,
A. Yokota et al. : Reactivity of glyoxylate and H 2 O 2 in glycolate pathway and vs shows how much H z O 2 is attacked by catalase. Therefore, v8 is the product of the activity of catalase (corresponding to k 1 or k 4 in the calculation by Chance et al. (1979)) and the concentration of H202. The molar concentrations of the intermediates in a cell were calculated using the cell volume reported by Giersch etal. (1980), 250 g l m g ~ chlorophyll (Chl), assuming that the plant cell including the central vacuole is homogeneous, or 50 I.tl mg -~ Chl excluding the vacuole; in the former case 1 mol rag- ~ Chl in a plant cell corresponds to 4. J 0 3 o r 2 . ] 0 4 mol 1-1, respectively, in concentration. The simulation of the glycolate pathway assumed that the influx rate of glycotate into the pathway from chloroplasts, vl, was constant, and that the concentrations of the substrates which are not intermediates of the glycolate pathway (glutamate or alanine for G G T and N A D H for hydroxypyruvate reductase) were high enough to drive the pathway at the maximal rate. Diffusion of the intermediates through organelle membranes was not considered. The enzyme reactions of the glycolate pathway were approximated by Michaelis-Menten equations. The reaction rate, vs, was represented by the Ping-Pong Bi Bi mechanism (Nakamura and Tolbcrt 1983). The conversion of glycine to serine was modeled as a single reaction in which K m for glycine and Vmax for the forward reaction are 1.0 mM and 100 gmol m g - ~ Chl h-~, respectively (Gardestr6m et al. 1980; Moore et al. 1977). Glycerate, formate, and CO 2 from glyoxylate and glycine were assumed to be end products not entering the glycolate pathway again. Thus, the change of the concentrations of the intermediates, A to I, is expressed by d[A]/dt
= v 1 -- v 2 ,
d[B]/dt
= v 2 -
d[C]/dt
= v 3 - v, + Vs,
u 3 - - I) 5 - - P 7 ,
d[D]/dt
= 0.5 "v4 -
d[E]/dt
= v s - v6,
d[F]/dt
= ~)6'
d[G]/dt
= v7,
d[H]/dt d[I]/dt
61
0
Glyoxylate [rnM) O.L 0.8 1.2 1.6 2.0 I
I
I
I
I
6 -6 E 3 c" 0
0 n
E 0 U
0
"ID c~
Cb)
40
o O~
T
"3 -i-., CJ
ct"
0
0
J , i I I 0.4 0.8 1.2 1.6 2.0 H202 (mM)
Fig. 2a, b. Reactivity of glyoxylate with H z O z . The reaction mixture consisted of 50 mM Hepes buffer (pH 8.0) and 1.28 mM H202 in (a) and 10 mM glyoxylate in (b). The reaction temperature was 25 ~ C
vs ,
= 0.5" v4, and = vz - v 7- vs .
The calculation of these differential equations was performed by the 4th-order Runge-Kutta method.
Results and discussion
Reactivity of glyoxylate and H202 . The stoichiometry of the reaction between glyoxylate and H 2 0 2 was confirmed. The reaction mixture (2 ml) consisted of 50 m M 4-(2-hydroxyethyl)-l-piperazineethane-sulfonic acid (Hepes)-KOH buffer (pH 7.0), 3.7 m M [1-14C]glyoxylate (2.05. 102 Bq I.tmol -~) and 5 m M H 2 0 z. The reaction was conducted for 30 min at 25 ~ C, and stopped by applying the reaction mixture to a column of Dowex 50 for the separation of glyoxylic and formic anions from the sodium ion, or by adding 0.2 ml of 1 M H z S O 4 to the mixture for determining H20 2 and evolved CO 2 . The consumption of 7.4 lamol of glyoxylate was accompanied by that of 7.56 gmol of H202 and the productions of 7.38 I.tmol of CO 2 and 7.05 I.tmol of formate. In
this reaction, the radioactivity of [1-14C]glyoxylate was recovered as CO 2 evolved, but not in formate, indicating that CO z was exclusively derived from the carboxyl carbon of glyoxylate, and formate from the aldehyde carbon. These results agree with those of earlier studies on the stoichiometry of the reaction (Grodzinski 1979; Halliwell and Butt 1974; Tolbert et al. 1949; Zelitich and Ochoa 1953; Zelitch 1972). Figure 2 shows the dependency of the reaction of glyoxylate and H 2 O 2 o n the concentrations of glyoxylate (a) and H 2 0 2 (b). In these cases, straight lines were obtained at concentrations below 10 raM. The results indicate that the reaction between glyoxylate and H202 is second-order. The rate constant of the reaction was 2.27 1 m o l - i s- 1 at pH 8.0 and 25 ~ C. The reaction of glyoxylate and H 2 0 2 w a s strictly dependent on the pH of the reaction mixture (Fig. 3). It did not proceed below pH 5.5. Above this pH, the rate constant of the reaction increased with increasing pH at least up to pH 8.3. At pH 7.0, the rate constant was 1.3 l mol - I s -1. Hatcher and Holden (1925) reported that glyoxylate was decomposed by H20 2 at acidic pH, but they used extremely high concentrations of glyoxylate and H 2 0 2 , unlike ours. The reaction of the unhydrated form of alde-
62
A. Yokota et al. : Reactivity of glyoxylate and
4
/
Q
~ 0-/,
%2
I-t202 in glycolate pathway
o
I
E
3.3
/
.--~1
0 -
I
6.0
7,0 pH
2-
,
8-0
Fig. 3. Effect of pH on the rate constant. The reaction mixture contained Hepes-KOH (o), 3-(N-morpholino)propanesulfonic acid (n), 2-(N-morpholino)ethenesulfonic acid (z~) or sodium acetate buffer (o), all at 50 mM, 5 mM glyoxylate and 1.28 m M H z O z . The reaction temperature was 25 ~ C
1I 15
] 20
] 25
Temperature
hyde with the peroxide anion is the Baeyer-Villiger faction (see e.g. Hassall 1957); divalent metal cations stimulate it. Since the hydration constant (unhydrated/hydrated) of glyoxylate at neutral pH is 1.6.10 -2 (Kfita and Valenta 1963) and the ionization constant of H 2 0 2 is 11.6 (Everett and Minkoff 1953), the exact concentrations of unhydrated glyoxylate and hydrogen peroxide anion are much lower at neutral pH than in the reaction mixture. At an acidic pH as used by Hatcher and Holden (1925), the hydration constant of glyoxylate decreases to the order of 10 -4, and H 2 0 2 is seldom or never ionized. Consequently, the reaction of glyoxylate and H202 requires very high concentrations of both compounds at acidic pH. Figure 4 shows the effects of temperature on the rate constant and the Arrhenius plots of the reaction. The rate constant at 35~ and pH 8.0 was extrapolated to be twice that at 25~ and the same pH. The slope of the line of the Arrhenius plots is the activation energy of the reaction, 68.66 kJ mol-1. In Table 1 are shown the effects of metal ions on the rate constant of the reaction of glyoxylate and H 2 0 2 a t pH 8.0 and 25 ~ C. Of the ions examined, Z n 2 + was most stimulative, and Fe z+ slightly stimulated the reaction. The metal ions at pH 7.0 had similar effects. Taking into account the stoichiometry of the reaction of glyoxylate and H 2 0 2 and the fact that the reaction was stimulated by Z n 2 + (Table 1), we consider the reaction mechanism of glyoxylate and H 2 0 2 to be that shown in Fig. 5. Glyoxylate is a mixture of the hydrated and unhydrated forms in equilibrium at physiological pH. In the presence o f Z n 2 +, the aldehyde carbon of glyoxylate easily
I 30
I 35
(~
Fig. 4. Effect of temperature on the rate constant. The reaction mixture was composed of 50 m M Hepes-KOH buffer (pH 8.0), 5 mM glyoxylate and 1.09 m M H20 2. I n s e t is an Arrhenius plot of the relationship between temperature and the rate constant
T a b l e 1. Effects of metal ions on the reaction of glyoxylate
with H202 . The reaction mixture consisted of 50 mM HepesKOH buffer (pH 8.0), 5 m M glyoxylate, 1.09 mM H20 z, and salts as shown in the table. The reaction temperature was 25 ~ C Salt
Rate constant (lmol - j s -1)
None KC1 (1 mM) NaC1 (1 mM) NH4C1 (1 mM) MgC12 (1 mM) CaC12 (1 mM) ZnC12 (1 raM) ZnC12 (0.1 raM) FeSO4 (0.1 raM) Fez(SO4) 3 (0.1 mM) MnC12 (0.1 raM)
2.27 2.21 2.09 2.09 2.49 2.88 9.99 3.65 4.00 2.72 2.31
donates its electron to the aldehyde oxygen and is nucleophilically attacked by the hydrogen-peroxide anion. The bond of the aldehyde and carboxyl carbons of the complex of glyoxylate and the peroxide anion is split through electron displacement; the former becomes formate and the latter CO 2 .
Simulation of the glycolate pathway of C3 plants. Purified GGT is three times as active on alanine as on glutamate (Noguchi and Hayashi 1981). Figure 6 represents the changes of concentrations of intermediates of the glycolate pathway when the
H202 in
A. Yokota et al. : Reactivity of glyoxylate and
glycolate pathway
Zn2+ ,'
OH 00 0I I III H--C--'C=0 T H - - C - - ' C = 0
I OH
Zn2 +
\
.._
0
0
I I
-~H-- C--'C'- 0 +
HZo Zn2+ j';',
-
=
00H000 Zn2+ ? 0 \ .,, , II ~'-~H--C'7":C-----0-'
P l a n t a 9 Springer-Verlag1985
Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena* A. Yokota, S. Kitaoka, K. Miura and A. Wadano Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 591, Japan
Abstract. The nonenzymatic reaction of glyoxylate and H202 was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H202 was secondorder, with a rate constant of 2.271 tool-1 s-1 at pH 8.0 and 25 ~ C. The rate constant increased by 4.4 times in the presence of Zn 2 + and doubled at 35 ~ C. We propose a mechanism for the reaction between glyoxylate and HzO 2 . From a comparison of the rates of H202 decomposition by catalase and the reaction with glyoxylate, we conclude that H202 produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO2 originates from glycine, but not from glyoxylate, in C 3 plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO 2 . In the simulation of the glycolate pathway of E u g l e n a , the rate constant was high enough to ensure the decarboxylation of glyoxylate by H202 to produce photorespiratory C O 2 during the glycolate metabolism of this organism. Key words: C 3 plant - E u g l e n a - Glycolate pathway (simulation) - Glyoxylate. * This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982)
Abbreviations. Chl = chlorophyll; GGT = glutamate: glyoxylate aminotransferase (EC2.6.1.4); Hepes=4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; SGT = serine: glyoxylate aminotransferase (EC 2.6.1.45)
Introduction
Glycolate, the substrate of photorespiration in C 3 plants, is produced through the action of ribulose1,5-bisphosphate carboxylase/oxygenase and phosphoglycolate phosphatase in chloroplasts, transferred to peroxisomes, and oxidized by glycolate oxidase (EC 1.1.3.1; for a review, see Tolbert 1980). The product of the oxidation, glyoxylate, may take part in one of the following three reactions; re-reduction to glycolate by the glycolateglyoxylate shuttle (Tolbert et al. 1970); decarboxylation by H20 2 in chloroplasts (Zelitch 1972) andor peroxisomes (Halliwell and Butt 1974; Grodzinski 1978); and transamination to glycine in peroxisomes (Rehfeld and Tolbert 1972). The existence of the glycolate-glyoxylate shuttle is obscured in C 3 plants because of the large difference of the pH optimum of chloroplast NADPH: glyoxylate reductase from the stromal pH under illumination and the low concentration of glyoxylate in situ (Schnarrenberger and Fock 1976). The oxidative decarboxylation of glyoxylate by H20 2 has received special attention in the metabolic elucidation of the evolution of photorespiratory CO 2 (Halliwell 1981; Zelitch 1971), but the occurrence of glutamate: glyoxylate aminotransferase (GGT) in peroxisomes and the rapid oxidation of glycine in mitochondria have tended to deny the involvement of non-enzymic glyoxylate decarboxylation in photorespiration (Chollett 1977; Ogren 1984). How glyoxylate escapes from oxidative degradation by H20 2 in peroxisomes has nor been elucidated (Tolbert 1980) because of lack of precise information on the reactivity of glyoxylate and H202, which has not been studied except for the
60
A. Yokota et al. : Reactivity of glyoxylate and HzO 2 in glycolate pathway
glycolate (A)
glycerate (F)
~
glycolate (A)
v2 hydroxypyruvate {El
O2 - ~ 8 H 2 0
+
1/2 02
v H202
"~"-~ glyoxylate (B)" v7
~
CO2 (G) + formate
NH2-d~176 v3
g l y /
Vcin~ ,cl
serine (D)
e (D)
~'~4
Perox isome
glycine (C)
CO2 (H) + NH3
itochondrion report of Hatcher and Holden (1925) who used extremely high concentrations of glyoxylate and H20 2 , and non-physiological pH's. Euglena gracilis, a photosynthetic protozoon, decarboxylates the carboxyl carbon of glycolate with no conversion to glycine during glycolate metabolism under highly photorespiratory conditions (Yokota and Kitaoka 1982). Glyoxylate formed by glycolate dehydrogenase (EC 1.1.99.14) in mitochondria may be decarboxylated in chloroplasts by H20 2 produced by a Mn 2 +-dependent N A D P H oxidase of chloroplasts (Yokota et al. 1983). We have measured the rate constant of the reaction between glyoxylate and H202, and simulated the glycolate-pathway kinetics of C 3 plants and Euglena using the rate constant we determined and published information. Our objective was to re-evaluate and possibly confirm the importance of the glyoxylate decarboxylation by H202 in the glycolate pathway of C 3 plants and Euglena. Materials and methods Materials. Glyoxylate was purchased from Sigma Chemical Co. (St. Louis, Mo., USA) and U 2 0 z from Nakarai Chemicals Co. (Kyoto, Japan). The concentration of glyoxylate was determined by measuring the oxidation of NADH with lactate dehydrogenase fromyeast and that of H20 z by light absorbancy
Fig. 1. Metabolic pathway of glycolate in C3-plant cells. Stoichiometries of the reactions are not taken into account in this figure. Kinetic parameters used in the simulation are as follows in the order: name of the metabolic step, maximum rate of the enzymatic reaction in gmol rag- 1 Chl h - 1, Km value for the substrate in mM: rate of the glycolate influx, vl, 60 (Zelitch 1971); glycolate oxidase, v2, 100, 0.38 for glycolate (Zelitch 1955; 1971); GGT, v3, 30, 1.2 for glyoxylate (Noguchi and Hayashi 1981; Yamazaki and Tolbert 1970; Zelitch 1971); alanine:glyoxylate aminotransferase, v3, 95, 1.2 for glyoxylate (Noguchi and Hayashi 1981); conversion of glycine to serine, v4, 100, 1.0 for glycine (Gardestr6m et al. 1980; Moore et al. 1977); SGT, vs, 20, 2.72 for serine, 0.15 for glyoxylate (Noguchi and Hayashi 1980); hydroxypyruvate reductase, v6, 80, 0.12 for hydroxypyruvate (Tolbert 1971; Tolbert et al. 1970). The estimations of the maximum reaction rates assumed 1.5 mg Chl = 1 g fresh weight =0.5 d i n / = 3 mg nitrogen = 20 mg protein, according to Zelitch (1971)
(McEwen 1971) and by the method of Hosoya and Morrison 1967) using peroxidase. Assay of reaction of glyoxylate and H202 . The reaction of glyoxylate and H202 was followed by measuring the initial decrease of light absorbance of H20 z (within 2 s). The extinction coefficient of H/O 2 at this wavelength is 61 1 mol-1 cm- t according to McEwen (197J). Since glyoxylate also absorbs light at 240 nm and the extinction coefficient of glyoxylate at this wavelength was 141mol - I cm -x, the apparent decrease of the absorbance at 240 nm was divided by 1.23 to give the actual decrease of the absorbance of H20 z in the reaction mixture. The decrease of the absorbance at 240 nm was followed with a recording spectrophotometer (model 200-100; Hitachi, Tokyo, Japan). Separation and determination of reactants and reaction products. The 14C02evolved from [l-l*C]glyoxylate was determined by the method of Yokota et al. (1978). After the reaction, the mixture was passed through a column (0.8 cm diameter, 2 cm long) of Dowex 50 (H+-type: BioRad Laboratories, Tokyo, Japan) to obtain the free-acid forms of the reactants and reaction products. Formic and glyoxylic acids were separated from each other by high-performance liquid chromatography (HPLC) with a Shodex C8t I column (Showa Denko, Japan) as reported in Yokota et al. (1983), and the recovery of formate was the same as that of glyoxylate.
Kinetic model Figure 1 shows the glycolate pathway with the intermediates, reaction rates and kinetic parameters which have been reported. v 1 is the rate of the influx of glycolate from chloroplasts into the glycolate pathway, v2 to v6 represent enzymatic reaction rates, v: is the reaction velocity between glyoxylate and HzO 2,
A. Yokota et al. : Reactivity of glyoxylate and H 2 O 2 in glycolate pathway and vs shows how much H z O 2 is attacked by catalase. Therefore, v8 is the product of the activity of catalase (corresponding to k 1 or k 4 in the calculation by Chance et al. (1979)) and the concentration of H202. The molar concentrations of the intermediates in a cell were calculated using the cell volume reported by Giersch etal. (1980), 250 g l m g ~ chlorophyll (Chl), assuming that the plant cell including the central vacuole is homogeneous, or 50 I.tl mg -~ Chl excluding the vacuole; in the former case 1 mol rag- ~ Chl in a plant cell corresponds to 4. J 0 3 o r 2 . ] 0 4 mol 1-1, respectively, in concentration. The simulation of the glycolate pathway assumed that the influx rate of glycotate into the pathway from chloroplasts, vl, was constant, and that the concentrations of the substrates which are not intermediates of the glycolate pathway (glutamate or alanine for G G T and N A D H for hydroxypyruvate reductase) were high enough to drive the pathway at the maximal rate. Diffusion of the intermediates through organelle membranes was not considered. The enzyme reactions of the glycolate pathway were approximated by Michaelis-Menten equations. The reaction rate, vs, was represented by the Ping-Pong Bi Bi mechanism (Nakamura and Tolbcrt 1983). The conversion of glycine to serine was modeled as a single reaction in which K m for glycine and Vmax for the forward reaction are 1.0 mM and 100 gmol m g - ~ Chl h-~, respectively (Gardestr6m et al. 1980; Moore et al. 1977). Glycerate, formate, and CO 2 from glyoxylate and glycine were assumed to be end products not entering the glycolate pathway again. Thus, the change of the concentrations of the intermediates, A to I, is expressed by d[A]/dt
= v 1 -- v 2 ,
d[B]/dt
= v 2 -
d[C]/dt
= v 3 - v, + Vs,
u 3 - - I) 5 - - P 7 ,
d[D]/dt
= 0.5 "v4 -
d[E]/dt
= v s - v6,
d[F]/dt
= ~)6'
d[G]/dt
= v7,
d[H]/dt d[I]/dt
61
0
Glyoxylate [rnM) O.L 0.8 1.2 1.6 2.0 I
I
I
I
I
6 -6 E 3 c" 0
0 n
E 0 U
0
"ID c~
Cb)
40
o O~
T
"3 -i-., CJ
ct"
0
0
J , i I I 0.4 0.8 1.2 1.6 2.0 H202 (mM)
Fig. 2a, b. Reactivity of glyoxylate with H z O z . The reaction mixture consisted of 50 mM Hepes buffer (pH 8.0) and 1.28 mM H202 in (a) and 10 mM glyoxylate in (b). The reaction temperature was 25 ~ C
vs ,
= 0.5" v4, and = vz - v 7- vs .
The calculation of these differential equations was performed by the 4th-order Runge-Kutta method.
Results and discussion
Reactivity of glyoxylate and H202 . The stoichiometry of the reaction between glyoxylate and H 2 0 2 was confirmed. The reaction mixture (2 ml) consisted of 50 m M 4-(2-hydroxyethyl)-l-piperazineethane-sulfonic acid (Hepes)-KOH buffer (pH 7.0), 3.7 m M [1-14C]glyoxylate (2.05. 102 Bq I.tmol -~) and 5 m M H 2 0 z. The reaction was conducted for 30 min at 25 ~ C, and stopped by applying the reaction mixture to a column of Dowex 50 for the separation of glyoxylic and formic anions from the sodium ion, or by adding 0.2 ml of 1 M H z S O 4 to the mixture for determining H20 2 and evolved CO 2 . The consumption of 7.4 lamol of glyoxylate was accompanied by that of 7.56 gmol of H202 and the productions of 7.38 I.tmol of CO 2 and 7.05 I.tmol of formate. In
this reaction, the radioactivity of [1-14C]glyoxylate was recovered as CO 2 evolved, but not in formate, indicating that CO z was exclusively derived from the carboxyl carbon of glyoxylate, and formate from the aldehyde carbon. These results agree with those of earlier studies on the stoichiometry of the reaction (Grodzinski 1979; Halliwell and Butt 1974; Tolbert et al. 1949; Zelitich and Ochoa 1953; Zelitch 1972). Figure 2 shows the dependency of the reaction of glyoxylate and H 2 O 2 o n the concentrations of glyoxylate (a) and H 2 0 2 (b). In these cases, straight lines were obtained at concentrations below 10 raM. The results indicate that the reaction between glyoxylate and H202 is second-order. The rate constant of the reaction was 2.27 1 m o l - i s- 1 at pH 8.0 and 25 ~ C. The reaction of glyoxylate and H 2 0 2 w a s strictly dependent on the pH of the reaction mixture (Fig. 3). It did not proceed below pH 5.5. Above this pH, the rate constant of the reaction increased with increasing pH at least up to pH 8.3. At pH 7.0, the rate constant was 1.3 l mol - I s -1. Hatcher and Holden (1925) reported that glyoxylate was decomposed by H20 2 at acidic pH, but they used extremely high concentrations of glyoxylate and H 2 0 2 , unlike ours. The reaction of the unhydrated form of alde-
62
A. Yokota et al. : Reactivity of glyoxylate and
4
/
Q
~ 0-/,
%2
I-t202 in glycolate pathway
o
I
E
3.3
/
.--~1
0 -
I
6.0
7,0 pH
2-
,
8-0
Fig. 3. Effect of pH on the rate constant. The reaction mixture contained Hepes-KOH (o), 3-(N-morpholino)propanesulfonic acid (n), 2-(N-morpholino)ethenesulfonic acid (z~) or sodium acetate buffer (o), all at 50 mM, 5 mM glyoxylate and 1.28 m M H z O z . The reaction temperature was 25 ~ C
1I 15
] 20
] 25
Temperature
hyde with the peroxide anion is the Baeyer-Villiger faction (see e.g. Hassall 1957); divalent metal cations stimulate it. Since the hydration constant (unhydrated/hydrated) of glyoxylate at neutral pH is 1.6.10 -2 (Kfita and Valenta 1963) and the ionization constant of H 2 0 2 is 11.6 (Everett and Minkoff 1953), the exact concentrations of unhydrated glyoxylate and hydrogen peroxide anion are much lower at neutral pH than in the reaction mixture. At an acidic pH as used by Hatcher and Holden (1925), the hydration constant of glyoxylate decreases to the order of 10 -4, and H 2 0 2 is seldom or never ionized. Consequently, the reaction of glyoxylate and H202 requires very high concentrations of both compounds at acidic pH. Figure 4 shows the effects of temperature on the rate constant and the Arrhenius plots of the reaction. The rate constant at 35~ and pH 8.0 was extrapolated to be twice that at 25~ and the same pH. The slope of the line of the Arrhenius plots is the activation energy of the reaction, 68.66 kJ mol-1. In Table 1 are shown the effects of metal ions on the rate constant of the reaction of glyoxylate and H 2 0 2 a t pH 8.0 and 25 ~ C. Of the ions examined, Z n 2 + was most stimulative, and Fe z+ slightly stimulated the reaction. The metal ions at pH 7.0 had similar effects. Taking into account the stoichiometry of the reaction of glyoxylate and H 2 0 2 and the fact that the reaction was stimulated by Z n 2 + (Table 1), we consider the reaction mechanism of glyoxylate and H 2 0 2 to be that shown in Fig. 5. Glyoxylate is a mixture of the hydrated and unhydrated forms in equilibrium at physiological pH. In the presence o f Z n 2 +, the aldehyde carbon of glyoxylate easily
I 30
I 35
(~
Fig. 4. Effect of temperature on the rate constant. The reaction mixture was composed of 50 m M Hepes-KOH buffer (pH 8.0), 5 mM glyoxylate and 1.09 m M H20 2. I n s e t is an Arrhenius plot of the relationship between temperature and the rate constant
T a b l e 1. Effects of metal ions on the reaction of glyoxylate
with H202 . The reaction mixture consisted of 50 mM HepesKOH buffer (pH 8.0), 5 m M glyoxylate, 1.09 mM H20 z, and salts as shown in the table. The reaction temperature was 25 ~ C Salt
Rate constant (lmol - j s -1)
None KC1 (1 mM) NaC1 (1 mM) NH4C1 (1 mM) MgC12 (1 mM) CaC12 (1 mM) ZnC12 (1 raM) ZnC12 (0.1 raM) FeSO4 (0.1 raM) Fez(SO4) 3 (0.1 mM) MnC12 (0.1 raM)
2.27 2.21 2.09 2.09 2.49 2.88 9.99 3.65 4.00 2.72 2.31
donates its electron to the aldehyde oxygen and is nucleophilically attacked by the hydrogen-peroxide anion. The bond of the aldehyde and carboxyl carbons of the complex of glyoxylate and the peroxide anion is split through electron displacement; the former becomes formate and the latter CO 2 .
Simulation of the glycolate pathway of C3 plants. Purified GGT is three times as active on alanine as on glutamate (Noguchi and Hayashi 1981). Figure 6 represents the changes of concentrations of intermediates of the glycolate pathway when the
H202 in
A. Yokota et al. : Reactivity of glyoxylate and
glycolate pathway
Zn2+ ,'
OH 00 0I I III H--C--'C=0 T H - - C - - ' C = 0
I OH
Zn2 +
\
.._
0
0
I I
-~H-- C--'C'- 0 +
HZo Zn2+ j';',
-
=
00H000 Zn2+ ? 0 \ .,, , II ~'-~H--C'7":C-----0-'
