Tautomerization of oxaloacetate and inhibition of maize leaf phosphoenolpyruvate carboxylase

Tautomerization of oxaloacetate and inhibition of maize leaf phosphoenolpyruvate carboxylase

Phytochemistry, Vol. 30, No. 3, pp. 751-756,1991 Printed in Great Britain. 0031-9422/91 53.00+0.00 Pergamon Press plc TAUTOMERIZATION OF OXALOACETAT...

662KB Sizes 0 Downloads 55 Views

Phytochemistry, Vol. 30, No. 3, pp. 751-756,1991 Printed in Great Britain.

0031-9422/91 53.00+0.00 Pergamon Press plc

TAUTOMERIZATION OF OXALOACETATE AND INHIBITION MAIZE LEAF PHOSPHOENOLPYRUVATE CARBOXYLASE GRIFFIN

Department

of Botany,

and

H. WALKER*

Institute of Biological

Chemistry,

GERALD

Washington

OF

E. EDWARDS

State University,

Pullman,

WA 99164-4230,

U.S.A.

(Received 11 June 1990)

Key Word Index-Zea

muys; Gramineae;

maize; oxaloacetate;

phosphoenolpyruvate

carboxylase..

Abstraet-Oxaloacetate is a p-keto acid which can readily undergo tautomerization to the enol form or non-enzymatic decarboxylation to pyruvate. Glycerol, histidine and glycine stimulate maize leaf P-enolpyruvate carboxylase activity (EC 4.1.1.31), and reduce the formation of the Mg-enol complex of oxaloacetate during catalysis. Oxaloacetate is a competitive inhibitor of maize P-enolpyruvate carboxylase. However, with Tris buffer inhibition is mixed and it causes an increase in the Mg-enol oxaloacetate complex. The inhibition of activity by oxaloacetate at pH 8.4 with 10 mM MgCl, (Ki = 135 PM) decreases significantly in the presence of 5 mM of the amines glycine, histidine, or hydroxylamine, but remains competitive. At low pH, malate inhibits maize P-enolpyruvate carboxylase and causes an increase in the Mg-enol tautomer of oxaloacetate. Inhibition of P-enolpyruvate carboxylase by oxaloacetate is suggested to be due to the Mg-enol complex. Certain effecters may influence the rate of catalysis through an effect on the quantity of the Mg-enol complex of oxaloacetate.

INTRODUCTION

Plants which use Crassulacean acid metabolism and C4 carbon fixation pathways for photosynthesis depend upon P-enolpyruvate carboxylase (EC 4.1.1.3 1) for initial fixation of atmospheric CO*, which results in synthesis of the keto form of oxaloacetate and orthophosphate from HCO; and P-enolpyruvate. This form of oxaloacetate is then reduced to malate, which serves as a donor of carbon to the reductive pentose phosphate pathway [l]. There are other possible fates of oxaloacetate which may either influence the efficiency of this process or regulate it. In solution oxaloacetate can rapidly form and maintain equilibrium mixtures between its keto and enol tautomers and between their hydrated forms. In addition, changes in pH, amine and cation concentrations, or solvent polarity, can influence the nonenzymatic decarboxylation of the keto form of oxaloacetate to pyruvate and CO2 through a metal enol form of the monocarboxylic acid [Z]. The ability of oxaloacetate to exist in different forms and to nonenzymatically decarboxylate may influence the activity of enzymes in which it is a substrate or a product. These have been important factors in the characterization of oxaloacetate tautomerase c31, oxaloacetate decarboxylase [4, 51, aspartate aminotransferase [6] and the triphasic kinetics of MDH (malate dehydrogenase) (since only the keto form serves as substrate) [7]. Relative to this latter study, the assay of P-enolpyruvate carboxylase coupled to MDH and NADH can result in underestimation of activity if the keto form of oxaloacetate is partly converted to the Mg-enol complex [8].

Non-enzymatic tautomerization of oxaloacetate can occur with widely varying rates, depending on conditions. This was shown by Pogson and Wolfe [7] in studies of the equilibrium between keto, enol and hydrated forms of oxaloacetate in the presence of MDH and NADH. Using a stopped flow technique, they showed that the time course of oxaloacetate reduction was triphasic. The first phase of NADH oxidation, ca 50 msec, was dependent on enzyme concentration and was attributed to the keto form. The slower phases of 1 and 10 set were independent of enzyme concentration, obeyed first order kinetics, and were due, respectively, to conversions from the enolated gem-diol to the keto form, and from one or both enolated species to the keto form. Changes in pH, metal concentrations, ionic strength and solvent polarity can increase this slower phase to min or hr [2, 93. We have considered tautomerization of oxaloacetate and the effects of oxaloacetate, malate, pH, Mg’+, glycerol, buffers and three amines (Gly, His, hydroxylamine) on the activity of maize leaf P-enolpyruvate carboxylase. RESULTS AND DISCUSSION

The difference in absorbance of a solution of oxaloacetate (0.1 mM) in 25 mM Hepes [4-(2_hydroxyethyl)-lpiperazineethanesulphonic acid] (pH 7.8) with and without the indicated additions of MnCl,, glycine or glycerol is shown in Fig. 1. Studies involving the rates of oxaloacetate tautomerization have shown that the keto form of oxaloacetate in aqueous solutions exhibits a broad absorption band centred at 255 nm [6], as is seen in the control (Fig. 1). With addition of glycine or glycerol, there *Current address: Chemistry and Metabolism, BASF is a large increase in absorbance and shift in maximum Corporation, Agricultural Research Center, P.O. Box 13528, absorption from 255 to 265 nm, indicating tautomerizResearch Triangle Park, NC 27709-3528,U.S.A. ation from the keto to the enol form. The addition of PHY 30:3-c

751

752

G. H. WALKER and G. E. EDWARDS

Wavelength

(nm)

Fig. 1. UV spectra of 0.1 mM solutions of oxaloacetic acid in the presence of 25 mM Hepes, pH 8.4, (control), or with the indicated additions. A measurements were recorded immediately upon addition of oxaloacetate to sample cuvette in the split beam mode.

MnCl, or MgCl, (not shown) shifts the absorption maximum to 285 nm, due to the formation of a metalenol-oxaloacetate complex (see [9] for different forms of oxaloacetate). The enol form (maximum absorbance at 265 nm) and the Mg-enol form (maximum absorbance at 285 nm) have much larger extinction coefficients than the keto form (maximum absorbance at 255 nm), which accounts for the much greater absorption upon addition of glycine, glycerol, or MnCl, (see Experimental for extinction coefficients). In the case of solutions containing MnCl,, there was a time dependent absorbance decrease due to the non-enzymatic decarboxylation of oxaloacetate to pyruvate. The control solutions and those containing glycine or glycerol remained stable for at least 2.5 hr (data not shown). The magnitude of the shift to the enol form upon addition of glycerol (Fig. I), is dependent on the glycerol concentration (Fig. 2). As the percentage of glycerol increases, the polarity of the solution decreases and conditions become more favorable for a shift to enol tautomerization. This can be explained by the fact that a decrease in the dielectric constant of the solvent causes a much greater stability of the enol complexes (by two orders of magnitude) than that of the keto form (ca lo-fold) [2]. This effect has been of particular interest to a number of investigators because enzyme active sites are often surrounded by hydrophobic groups [2]. The formation of the Mg-enol complex depends on the ratio of Mg/oxaloacetate. Preliminary experiments with HEPES buffer (pH 7.8 or 8.4) indicated maximum formation of this complex with a ratio of Mg/oxaloacetate of 500-1000 (based on the A285-A265, also see [lo]). Under conditions giving a ratio of Mg/oxaloacetate of 1000, glycerol (25% v/v), histidine (5 mM) and glycine (5 mM) cause a shift from the Mg-enol oxaloacetate complex (285 nm absorbance) towards the free enol form (265 nm) (Fig. 3). These additions had their maximum effects (decrease in the Aze5- A,,,) with increasing pH values, and the effect of histidine was the most pronounced of the two amines. At the most acidic pH value (7.2), the least difference between the control and effecters was observed. This pH dependent difference between the two chromophores may be explained by the previous findings of

6-

5-

I 5

25 mM Hepes, pH 8.4 I I IO 15

% Glycerol

I 20

(V/V)

Fig. 2. Absorbance changes of 0.1 mM oxaloacetic 265 nm with 25 mM HEPES, pH 8.4, with increasing content.

acid at glycerol

Steinhaus et al. [ll]. These authors have suggested that, in the absence of either histidine or glycine, the major fraction of oxaloacetate is in the form of an inactive dinuclear complex. This results in liberation of substantial amounts of uncombined oxaloacetate, which forms metal-oxaloacetate complexes. The difference in effect between the two amines is probably due to histidine having an imidazole group. This group can act as a conjugated base with a pK, = 8.0, and it keeps the 285 nm chromophore from becoming a stable and inactive hydrate through base catalysis [2, 121. Glycerol is less effective than histidine in preventing the formation of the Mg-enol complex (the 285 nm chromophore), probably due to its inability to keep the hydrated form of the enol complex from forming (Fig. 3A). An example of the effects of these substances on the equilibrium and rate constants

Tautomerization I

I

7.5

7.6

I

I

-

(a) 0.1 mM OAA

o,m_

IOOmM Mg Cl, 0.05-

0.04-

0.03-

0.02-

O.Ol0

7.2

7.3

7.4

7.7

70

PH

I

40-

(b) 5mM Mg Cl,

,,o__I’

o----o----q o----o---_o___+5mM

HIS

30-

20-

IO-

7.6

7.0

i30

8.2

8.4

of oxaloacetate

5 mM histidine, remaining constant between pH 7.8 and 8.4 (39.5 ymol min- ’ mg- 1 protein). Reactions containing 25% glycerol and 5 mM glycine followed, respectively, in effectiveness. The control showed a decrease between pH 7.8 and pH 8.4, with a loss in activity of 5.5 pm01 min-’ mg-’ protein. The large stimulation of activity by the amines or glycerol at higher pH is also at a pH which is most favorable for reduction in the amounts of Mg-enol complex. Because the stimulation of activity by amines and glycerol may occur in part by prevention of formation of the Mg-enol oxaloacetate complex, the degree of tautomerization during the assay was determined at pH 7.2, 7.8 and 8.4 (Table 1). In one assay, the release of orthophosphate and the formation of Mg-enol oxaloacetate was determined. In another assay, P-enolpyruvate carboxylase was coupled to MDH and malate formation was determined by HPLC. No tautomerization was detected at pH 7.2 and there was excellent agreement between the Pi-release assay (7.8 pmol min- ’ mg- ’ protein) and the MDH coupled assay (7.7 pmol min-’ mg- ’ protein). At pH 7.2 glycine stimulated the activity by 16% and histidine by 28% (calculated from data using the Pi assay). This stimulation was accompanied by rates of pyruvate formation of 1.2 and 3.0 pmolmin-’ mg-’ protein for glycine and histidine, respectively. Under more alkaline conditions (pH 7.8), there was evidence of tautomerization. The rate of tautomerization to the Mg-enol complex averaged 9.4 pmol min- 1mg- 1 protein (reaching a concentration of 49 PM after 90 set). However, the 285 nm form did not begin to appear until after the reaction had progressed for 48-50 sec. This resulted in a maximum rate of14.1 ~molmin-lmg-l protein during the latter part of the assay (Table 1, parentheses). In the presence of histidine or glycine, no detectable amounts of the 285 nm form were found.

Table 1. Stimulation

PH

Fig. 3. (A) Changes between the 285 nm minus 265 nm chromophores of oxaloacetic acid (0.1 mM) at varying pH, with 100 mM MgCl, and with the indicated effecters. (B) Influence of various etfectors on maize P-enolpyruvate carboxylase activity assayed by Pi formation (see Experimental). In both experiments, the 1 ml reaction mixture contained 20 mM each of MES, Hepes and Tricine adjusted to the indicated pH values.

of maize P-enolpyruvate activity by amines

carboxylase

Specific activity @no1 min-’ mg-’ protein) PH

Addition

34Onm*

Pit

285 nmt

1.2

Control 3 mM Gly 3 mM His Control 3 mM Gly 3 mM His Control 3 mM Gly 3 mM His

7.1 8.6 10.5 27.4 28.5 34.8 26.2 30.8 35.2

7.8 8.6 10.0 31.4 33.0 36.5 25.8 31.5 35.7

NDS ND ND 9.4 (14.1)$ ND

7.8

can be given for pH 7.4. This pH value showed the highest rate constant for the control (7.5 x 10M4set-’ for Mgenol formation from the keto form) and a K,, for the keto to Mg-enol form of 2.1. The addition of 5 mM glycine decreased the rate constant to 6.2 x 10e4 see-’ and the K,, to 1.6, the addition of 25% glycerol decreased the rate constant to 2.2 x 10e4 set-’ and the K,, to 0.6, while addition of histidine decreased the rate constant to 2.2 x 10m4 set- ’ and the K,, to 0.05. The influence of glycerol, glycine and histidine, on the activity of PEP carboxylase was determined at varying pH in the presence of 5 mM MgClz (Fig. 3B). The activity in the presence of 5 mM histidine, 5 mM glycine and 25% glycerol was higher throughout the pH profile compared to the control. The highest activity was in the presence of

153

8.4

IL,, ND ND

*Assay mixture contained MDH and NADH with 3.5 ng enzyme ml-r (see Experimental). tin assay for determining Pi and organic acids (without NADH and MDH); the 3 ml reaction mixture (3.5 ng enzyme ml-r) was initiated by addition of 1.5 mM P-enolpyruvate. Pi was determined after 60 set and Mg-enol OAA (spectrophotometrically at 285 nm) after 90 sec. $ND =Not detected. §Maximum rate after 50 sec. IIRate calculated after 5 sec.

G. H. WALKERand G. E. EDWARDS

154

At pH 8.4, the control rates of P-enolpyruvate carboxylase activity were lower those at pH 7.8 (Pi assay). In the control at pH 8.4, a rate of formation of Mg-enol oxaloacetate of 11 pmolmin-‘mg-’ protein was observed in less than 5 set (resulting in a concentration of 3.2 PM), and thereafter, the rate gradually increased exponentially. In the presence of glycine or histidine, tautomerization was suppressed. The stimulation of enzyme activity by glycerol and amines may occur, in part, through prevention of formation of the Mg-enol complex of oxaloacetate, which is a suggested inhibitor of the enzyme. The effects of glycine, histidine and hydroxylamine on the oxaloacetate inhibition of P-enolpyruvate carboxylase activity were examined. The results from a series of experiments performed to calculate the K, for P-enolpyruvate and Ki for oxaloacetate with and without amines are shown in Table 2. Oxaloacetate acts as a competitive inhibitor, increasing the apparent K, for P-enolpyruvate without effecting the V,,,. With addition of amines (5 mM), the G., was unaffected, the K, values for oxaloacetate increased and the K, values for P-enolpyruvate in the presence of oxaloacetate approach those seen in the absence of oxaloacetate (control). The amines may have their effect through prevention of tautomerization of the oxaloacetate to a kinetically inactive and inhibitory metal-en01 complex. The nature of the pattern of inhibition of P-enolpyruvate carboxylase by oxaloacetate was also influenced by the buffer used. The results of oxaloacetate inhibition of maize P-enolpyruvate carboxylase activity with 50 mM Tris buffer (pH 8.4) are shown in Fig. 4A. Using a concentration of 0.5 mM oxaloacetate with increases in Penolpyruvate as indicated, the inhibition was mixed. There was a considerably different pattern of inhibition in a reaction medium containing 50 mM Hepes, pH 8.4 (Fig. 4B). The results of this experiment showed the inhibition by 0.5 mM oxaloacetate to be competitive with an increase in the K, for P-enolpyruvate from ca 0.8 to 1.6 mM. Experiments at lower pH, with Hepes buffer (pH 7.2 and 7.8), also showed competitive inhibition of Penolpyruvate carboxylase activity by 0.5 mM oxaloacetate (data not shown). Various reports in the literature indicate that oxaloacetate is an inhibitor of plant Penolpyruvate carboxylase [13-171, but there are discrepancies in the type of inhibition reported. For ex-

ample, Lowe and Slack [14] in assays at 50 mM Tris at pH 8.4, reported non-competitive inhibition of maize leaf P-enolpyruvate carboxylase by oxaloacetate. In contrast, Coombs et al. [15] in experiments using 50 mM HEPES buffer, pH 8, found competitive inhibition by oxaloacetate of P-enolpyruvate carboxylase from the tropical grass Pennisetum purpureum. Increasing concentrations of Tris buffer results in a dramatic shift in equilibrium from the kinetically active keto form to the more spectrophotometrically active Mg-enol form (Fig. 5). This effect of Tris buffer is caused by an increase in base catalysed tautomerization rate [12], which can cause discrepancies in enzyme assays which involve oxaloacetate [8]. Therefore, the mixed inhibition (Fig. 4A) may be due to Tris preventing conversion of the catalytic intermediate enol form of oxaloacetate to the keto form, or because it causes tautomerization of the keto form to an inhibitory enol form. It has previously been shown that inhibition of maize P-enolpyruvate carboxylase by malate or asparate is most pronounced at pH values between 7.0 and 7.5 [18, 191. This was also observed in the present study using the assay based on Pi formation (Fig. 6). In the presence of 10 mM malate at pH 7.0, P-enolpyruvate carboxylase activity was inhibited by ca 80%. With increasing pH values, the inhibition decreased to ca 50% (pH 8.0-8.4). Interestingly, the rate of production of the Mg-enol form of oxaloacetate in the presence of malate was 7.2 pmol min-‘mg-’ protein at pH 7.0, compared to only 1.5 ~molmin-l mg-’ protein at pH 8.4. This resulted in micromolar levels of Mg-enol oxaloacetate being formed during the assay in the presence of malate (e.g. ca 25 PM Mg-enol oxaloacetate at pH 7.0 after one min). Assay of the enzyme by coupling to MDH gave an even lower apparent activity at low pH in the presence of malate, which is likely due to the oxaloacetate enol-Mg complex not being recognized by MDH (data not shown). Addition of 5 mM glycine to the reaction medium reversed the inhibition by malate and resulted in a slight stimulation in enzyme activity. The formation of the oxaloacetate enol tautomer in the presence of 5 mm glycine was not observed under these conditions. Identical experiments substituting aspartate for malate showed a similar response by P-enolpyruvate carboxylase in terms of inhibition of enzyme activity and increased levels of the enol tautomer at low pH (data not shown).

Table 2. Changes in K, values for oxaloacetate (OAA) with maize leaf P-enolpyruvate carboxylase in the presence of 5 mM histidine, NH,OH or glycine at pH 8.4

Conditions*

Ki (OAA)? mM

K, (P-enolpyruvate) mM

V,,,., (pm01 min-’ mg-’ protein)

Control, Varying Varying Varying Varying

0.135 1.2 0.85 0.60

0.6 1.3 0.6 0.7 0.75

29 29 30 31 30

minus OAA OAA OAA + 5 mM His OAA + 5 mM NH,OH OAA + 5 mM Gly

*Assays were performed at 25” in a volume of 1 ml containing 25 mM Tricine-KOH (pH 8.4), 3.5 pg P-enolpyruvate carboxylase, 3 mM MgCI, and 10 mM KHCO,, and the indicated concentration of amines and 0, 0.25, 0.5, 0.75 and 1.0 mM oxaloacetate. After 2 min preincubation, reactions were initiated with 5 mM P-enolpyruvate and allowed to run for 2 min. Reactions were terminated with 10% HCIO, and assayed for Pi release (see Experimental). tKi values were determined using Dixon plots.

Tautomerization (a)

50mM pH 0.4

of oxaloacetate

755

Tris 0.16..

(b) 50mM pH 8.4

I/P-eno1pyruvote

Hepes

hhc’) 0

+OAA (0.5mM)

20

I 40

60

25 mM Hepes, pH 8.4 1 I 1 80 loo 120

Tris CmM)

Fig. 5. Absorbance changes of 0.1 mM oxaloacetic acid at 285 nm with increasing concentrations of Tris added to 25 mM Hepes, pH 8.4.

that the enol form is a strong competitive inhibitor of the reaction with respect to PEP [20]. EXPERIMENTAL -1.0

0 I/P-enolpyruvote

I.0

2.0

(mM-‘)

Fig. 4. Inhibition of purified maize leaf P-enolpyruvate carboxylase by oxaloacetate in the presence of Tris or Hepes as buffer (pH 8.4). The 1 ml reaction mixtures contained 3 mM MgCl,, 10 mM KHCO,, 3.5 pg P-enolpyruvate carboxylase and, as indicated, 0.5 mM oxaloacetate and either (A) 50 mM Tris or (B) 50 mM Hepes buffer at pH 8.4. Reactions were initiated, after a 1 minute preincubation with oxaloaeetate, by the addition of the indicated concentration of P-enolpyruvate, and allowed to run for 2 min at 27”. Reactions were terminated by addition of 0.5 ml 10% perchloric acid and assayed for free Pi (see Experimental).

Malate, which interferes with catalysis [16-191, may do so by inhibiting the conversion of the enol form (a catalytic intermediate) to the kinetically active keto form. Alternatively, magnesium, which is required for tautomerization of oxaloacetate from the enol to the keto form during catalysis, may be chelated by malate, favouring the accumulation of the enol form (M. O’Leary, personal communication). While the mechanism of malate inhibition is uncertain, the mechanism of inhibition may in-

volve the synthesis of Mg-enol oxaloacetate during maximum inhibition and tautomerization to the keto form at lower levels of inhibition. In summary, P-enolpyruvate carboxylase in plants may be regulated, in part, by concentrations of C4 dicarboxylic acids. Formation of the Mg-enol tautomer of oxaloacetate could have a role in this inhibition. As oxaloacetate can undergo tautomerization and non-enzymatic decarboxylation during the course of enzymatic reaction, it is difficult to fully characterize such inhibition. Thus, this has been further evaluated using diethyloxaloacetate, a stable analogue of oxaloacetate, with evidence

Plant material and partial purification of maize P-enolpyruvate carboxylase. Zea mays was grown for three weeks under a

controlled temp. and photoperiod of 27/22” and 14/10 hr as previously described [21, 221. P-enolpyruvate carboxylase was isolated from leaves using 25mM MOPS [3-(iV-morpholino)propanesulphonic] buffer (pH 6.8) according to the method of ref. [23]. The final 70% satd (NH&SO, ppt was desalted using a Sephadex G-25 column before fractionation on a DEAEcellulose column. The DEAE column was first eluted with 80mM, then 120mM KC1 in 25 mM MOPS (pH 6.8). Frs containing P-enolpyruvate carboxylase activity were pptd with 60% (NH&SO, and centrifuged at 15000 g for 15 min. The resulting pellet was resuspended in 10 mM MOPS (pH 6.8), 25% glycerol, 10mM MgCl,, 2 mM DTT and stored at -20”. Further purification of the protein was as previously described [21,22]. This involved using size exclusion HPLC, collecting frs containing the 400 000 tetramer of P-enolpyruvate carboxylase and recycling through the size exclusion column twice. The resulting frs showed 95% or greater purity as determined by SDS-gel electrophoresis. Protein measurement. Total protein concn was determined according to the method of ref. [24]. Assay of P-enolpyruvate carboxylase. The assay of the enzyme by determination of Pi release was according to the procedure of ref. [8], which is a modification of a procedure used in mitochondrial studies [25]. Unless otherwise specified, all reactions were run at 27”. The 1 ml reaction mixt. contained 3.5 pg P-enolpyruvate, 3 mM MgCl, and 10 mM KHCO, with the indicated concentrations of buffers and pH. In cases where P-enolpyruvate carboxylase was assayed by malate formation using HPLC (Table l), 4 IU of MDH and 1 mM NADH were included in the assay medium. Measurements of organic acids by HPLC. Sepn of organic acids was through a Bio-Rad guard column and an Aminex HPX-87 analytical column using a 15 mN H,PO, mobile phase at 30”. For detection and quantification, a UV detector with a Zn lamp and 214 nm filter was used. For additional details conceming quantification of organic acids, see previous refs [26, 271.

756

G. H. WALKERand G. E. EDWARDS

0

3 7.0

, 72

74

76

78

8.0

I

I

82

84

0 86

PH

Fig. 6. Effect of malate and malate plus glycine on the activity of P-enolpyruvate carboxylase at varying pH. The 1 ml reaction mixture contained 20 mM each of MES, Hepes, and Tricine, 10 mM KHCO,, 3.5 pg P-enolpyruvate carboxylase and 3 mM MgCl,. The enzyme was allowed to preincubate with the indicated concentrations of 10 mM malate, 5 mM glycine + 10 mM malate or no additions (control). Activity was measured as Pi release. The rate of formation of the Mg-enol tautomer of oxaloacetate (determined spectrophotometrically at 285 nm) is shown for the assay in the presence of 10 mM malate. Reactions were stopped by addition of an equal vol. (1 ml) of o-4” mobile phase containing 50% MeCN and then placing the solns on ice. Spectrophotometric detection of tautomeric forms of oxaloacetate. Reactions were monitored at the indicated wavelengths

in the split-beam mode or, where indicated, in the dual beammode monitoring the indicated wavelength minus Ashr,. Reaction vols (0.7 ml) were maintained at 27”. The extinction coefficient used for the oxaloacetate-keto tautomer was 8.0 x 10z M-i cm-’ at 255 nm, while for both the oxaloacetateenol tautomer at 265 nm and the oxaloacetate-enol metal complex at 285 nm the extinction coefficient used was 1.1 x lo4 M-* cm-’ [2, 63. Preparation of stock oxaloacetate solutions. Stock solns of oxaloacetate (3 mM) were prepd fr. daily. This was done by adding solid oxaloacetate to a cold, 25 mM Tricine-O.1 M KOH buffer soln (pH 9), then back titrating with 5 M HCI to pH 7 using a pH meter. This procedure was used to minimize the formation of oxaloacetate monoanion which decarboxylates more rapidly than oxaloacetate-H, or oxaloacetate*-. At the beginning of the expt, and several times throughout the day, HPLC was used to examine 5 nmol samples of stock soln of oxaloacetate in order to assess its stability.

6. Hess, J. L. and Reed, R. E. (1972) Arch. Biochem. Biophys. 153, 226.

7. Pogson, C. I. and Wolfe, R. G. (1972) Biochem. Biophys. Res. Commun. 46, 1048.

8. Walker, G. H., Ku, M.S.B. and Edwards, G. E. (1986) Arch. Biochem. Biophys. 248,489.

9. Covey, W. D. and Leussing, D. L. (1974) 1. Am. Chem. Sot. %, 3860.

10. Kosicki, G. W. and Lipovac, S. N. (1964) Can. J. Biochem. 42, 403.

11. Steinhaus, R. K., Raghavan, N. V. and Leussing, D. L. (1977) J. Inorg. Nucl. Chem. 39, 1871. 12. Emly, M. and Leussing, D. L. (1981) J. Am. Chem. Sot. 103, 628.

13. Ting, I. P. (1968) Plant Physiol. 43, 1919. 14. Lowe, J. and Slack, C. R. (1971) Biochem Biophys. Acta 235, 207.

15. Coombs, J., Baldry, C. W. and Bucke, C. (1973) PIanta 110, 95.

16. Bhagwat, A. S. and Sane, P. V. (1976) Indian J. Exp. Biol. 14, 155. 17. Raghavendra, A. S. and Das, V. S. R. (1975) Biochem. Biophys. Res. Commun. 66, 160.

18. Huber, S. C. and Edwards, G. E. (1975) Can. J. Botony 53,

Acknowledgement-This

research was supported Standard Oil Company of Ohio (Sohio).

by The

REFERENCES 1. Edwards, G. E. and Huber, S. C. (1981) in The Biochemistry of Plants Vol. 8 (Hatch, M. D. and Boardman, N. K., eds), pp. 238-281. Academic Press, New York. 2. Leussing, D. L. (1982) in Advances in Inorganic Biochemistry Vol. 5 (Eickhorn, G. L. and Marzilli, L. G., eds), pp. 171-200. Elsevier, New York. 3. Annette, R. G. and Kosicki, G. W. (1969) J. Biol. Chem. 244, 2059. 4. Wojtczak, A. B. and Walajtys, E. (1974) Biochem. Biophys. Acta. 347, 168. 5. Creighton, D. J. and Rose, I. A. (1976) J. Biol. Chem. 251,61.

1925.

19. Huber, S. C. and Sugiyama, T. (1986) Plant Physiol. 81,674. 20. Walker, G. H. and Edwards, G. E. (1990) Photosynth. Res. 25, 101. 21. Walker, G. H., Ku,.M. S. B. and Edwards, G. E. (1986) J. Liq. Chromatog. 9, 861.

22. Walker, G. H., Ku, M. S. B. and Edwards, G. E. (1986) Plant Physiol. SO, 848.

23. Uedan, K. and Sugiyama, T. (1976) Plant Physiol. 57, 906. 24. Bradford, M. M. (1976) Anal. Biochem. 72, 248. 25. Kapland, R. S. and Pedersen, P. L. (1983) Biochem. J. 212, 279.

26. Walker, G. H. and Oliver, D. I. (1983) Arch. Biochem. Biophys. 225, 847.

27. Oliver, D. J. and Walker, G. H. (1984) Plant Physiol. 76,409.