The substrate specificity, kinetics, and mechanism of glycerate-3-kinase from spinach leaves

ARWI\‘ES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 236, No. 1, January, pp. 185-194, 1985

The Substrate Specificity, Kinetics, and Mechanism Glycerate-3-kinase from Spinach Leaves’ LESZEK

Biochemistry

A. KLECZKOWSKI,’ AND WARREN Department,

University

DOUGLAS L. ZAHLER of Missouri,

of

D. RANDALL,

Columlria, Missouri

65211

Received May 25, 1984, and in revised form August 13, 1984

Glycerate-3-kinase (EC 2.7.1.31) from spinach leaves shows absolute specificity for D-glycerate as phosphate acceptor, yielding 3-phosphoglycerate as a product. ATP complexed with either Mg2+ or Mn2+ is the preferred phosphate donor. The enzyme has K, (D-glycerate) = 0.25 mM, Km (Mg-ATP) = 0.21 mM, V,,, = 300 pmol min-l mg protein-l, and a turnover number = 12,000 - min *. The equilibrium constant for the reaction is approximately 300 at pH 7.8. Pyrophosphate, 3-phosphoglycerate and ribulose 1,5-bisphosphate are the strongest inhibitors among the phosphorylated and nonphosphorylated metabolites tested; however, their regulatory role in &JO is questioned. Substrate kinetics, as well as product and analog inhibition data, are consistent with a sequential random mechanism. The distinct characteristic of the glycerate kinase-catalyzed reaction is the formation of a dead-end complex between tr I!% Academic Pwss.Inr. the enzyme, D-glycerate, and 3-phosphoglycerate.

metabolite of both pathways (1, 2, 5). As an exclusively chloroplast enzyme (5-7), glycerate kinase has been proposed to be regulated solely by substrate availability, since the most frequent modulators of stromal enzymes (light-dependent changes in energy charge, reducing compounds, and pH) have no effect on the activity of this enzyme (7). As we have previously reported (8), the enzyme purified to apparent homogeneity from spinach leaves is a monomer of 40,000 with a broad pH optimum (6.5-8.5) and with a p1 value of 4.8. Similar M,, pH optima, and p1 values were found for partially purified glycerate kinases from pea and rye leaves (7). Glycerate kinases have also been identified and studied in non-green plant tissues (9, lo), as well as in liver (11-13) and some bacteria (14-17). In liver, glycerate kinase is the key enzyme of gluconeogenesis from serine (12). The enzyme from rat liver mitochondria is a monomer of about M, 53,000 (12) and is specific for D-

Glycerate kinase is the terminal enzyme of the oxidative photosynthetic carbon pathway (photorespiration) in plants. This pathway accounts for a considerable reduction of the net photosynthetic rate in a number of species (1, 2). Among the postulated reactions of the photorespiratory pathway are the formation of glycerate from hydroxypyruvate in the peroxisomes, transport of glycerate across the peroxisomal and chloroplast membranes, and its subsequent phosphorylation in the stroma of chloroplasts (1, 3, 4). Glycerate kinase links the flow of photorespiratory carbon to the reductive photosynthetic carbon metabolism by the production of phosphoglyceric acid, which is the common ‘This research was supported in part hy NSF Grant PCM-8104659 and USDA/CRGO Grant 592291-1-1-366-1. This is Journal Report no. 9665 from Missouri State Agricultural Experiment Station. *To whom all correspondence should be addressed at 322A Chemistry Bldg., UMC, Columbia, MO. 65211. 185

0003-9861/85 $3.00 Copyright All rights

0 1985 by Academic PEW, Inc. of reproduction in any farm reserved.

186

KLECZKOWSKI,

RANDALL,

glycerate yielding, 2-PGA3 as a product of the reaction (13). In Escherichia coli, glycerate kinase participates in the socalled “glycerate pathway” (14-15). The enzyme was found to be specific for D stereoisomer of glycerate (14), yielding 3PGA as a product (15). Crystallized enzyme from the same source is very sensitive to hydroxymercuribenzoate and iodoacetate, suggesting that sulfhydryl groups may be involved in catalysis (15). The purpose of the present study was to characterize the homogenous enzyme from spinach leaves and to study the kinetic mechanism of the glycerate kinasecatalyzed reaction. To our knowledge this is the first kinetic analysis of glycerate kinase from any source. MATERIALS

AND

METHODS

Reagents. D, L, and DL-&Cerate, 3-PGA, 2-PGA, fructose 2,6-diphosphate, ADP, UTP, and GTP were obtained from Sigma. ATP, NADH, PEP, glycolate, and phosphoglycolate were from P-L Biochemicals, Inc. All other reagents were commercial preparations of the highest grade available. Glyceraldehyde phosphate dehydrogenase, pyruvate kinase, lactate debydrogenase, phosphoglycerate mutase (all from rabbit muscle), and phosphoglycerate phosphokinase, as well as enolase and hexokinase (all from yeast) and glucose-6-phosphate dehydrogenase (Leuconostoc mesentef-tides) were from Sigma. Pur$cation of glycerate kinase. Glycerate kinase was purified to homogeneity using the procedure of Kleczkowski and Randall (8) with slight modifications. Purification steps involved polyethylene glycol 8OOO/MgClz precipitation, DEAE-cellulose chromatography, molecular sieving on Sephadex G-75SF column, and dye-ligand affinity chromatography on Affi-Gel Blue (Bio-Rad) and Green A (Amicon) columns. Procedure modifications: The elution buffers

a Abbreviations used: PGA, phosphoglyceric acid; PEP, phosphoenolpyruvic acid; SDS, sodium dodecyl sulfate; PK, pyruvate kinase; LDH, lactate dehydrogenase; PGA-PK, phosphoglycerate phosphokinase; GAP-DH, glyceraldehyde phosphate dehydrogenase; HK, hexokinase; GGP-DH, glucose-6-phosphate dehydrogenase; DHAP, dihydroxyacetone phosphate; RuBP, ribulose bisphosphate; Mops, 4-morpholinepropane sulfonic acid; Tricine, N-[2-hydroxy-l,lbis(hydroxymethyl)ethyllglycine.

AND

ZAHLER

were degassed, Affi-Gel Blue column was equilibrated and eluted with 20 ITIM Mops, pH 6.75, 2 mM MgClz, and 14 mM 2-mercaptoethanol; elution was carried out with a 200-ml linear gradient of O-3 mM ATP. Degassing of buffers helped to maintain stability of glycerate kinase, especially during affinity dye-ligand steps. Modifications in the Affi-Gel Blue procedure (lowering the pH and an extension of ATP gradient) increased the yield and specific activity of glycerate kinase eluted from the column. Protein determination and the conditions of SDSpolyacrylamide electrophoresis were as previously reported (8). Assay of glycerate kinase. Assays were carried out at 25°C by monitoring NADH oxidation at 340 nm with a recording spectrophotometer. One unit of glycerate kinase activity was defined as the amount of enzyme required to oxidize 1 pmol NADH/min under conditions of 5 mM ATP, 10 mM MgClz, and 5 mM DL-glyCt?rate using the PK/LDH or PGA-PK/ GAP-DH coupling enzyme systems. All coupling enzymes were desalted on small Sephadex G-25 columns before use. PK/LDH system. The assay contained, in 1 ml, 0.1 M Tricine, pH 7.8, 2.5 mM PEP, 60 mM KCl, 0.2 mM NADH, varying amounts of glycerate kinase, and 5 units each of PK and LDH. The concentration of ATP, glycerate, and MgClz varied and is specified elsewhere. Reactions were initiated by addition of glycerate. PGA-PK/GAP-DH system. The assay contained, in 1 ml, 0.1 M Tricine, pH 7.8, 0.2 mM NADH, varying amounts of glycerate kinase, and 5 units each of PGA-PK and GAP-DH. The concentration of ATP, glycerate, and MgClz varied and is specified elsewhere. Reactions were initiated by addition of glycerate. Phosphoglycerate mutase/enolase/PK/LDH system. The assay contained, in 1 ml, 0.1 M Tricine, pH 7.8, 0.25 mM NADH, 60 mM KCI, varying amounts of glycerate kinase, and 6 units each of coupling enzymes. The concentration of ATP, glycerate, and MgClz varied and is specified elsewhere. Reactions were initiated by addition of glycerate. Specijicity of glycerate kinase for phosphate acceptor. Each of the following compounds was checked as a possible substrate for glycerate kinase: Dgluconate (20 mM), DL-glyceraldehyde (20 mM), Dglycerate (0.82 mM), L-glycerate (2.35 mM), glycerol (20 mM), glycolate (20 mM), D-kid&e (10 mM), DLmalate (20 mM), and DL-Serine (20 mM). All assays contained, in 1 ml, 0.1 unit glycerate kinase, 0.2 IIIM ATP, and 5.2 mM MgClz in PK/LDH system. Specijicity of glycerate kinase for phosphate donor. Assay of pyrophosphate-dependent rates. A 3-ml mixture of 0.1 M Tricine, pH 7.8, 5 mM MgClz, 5 mM DL-glycerate, 1 mM pyrophosphate, and 1 unit glycerate kinase was incubated for 5 min at 25’C, and

SPINACH

GLYCERATE

then heated at 70°C for 5 min. After cooling to 25°C 800 ~1 of incubation mixture was assayed in a l-ml final volume with 0.2 mM NADH, 60 mM KCI, and phosphoglycerate mutase/enolase/PK/LDH (6 units each). Reactions were initiated with 5 mM ADP. As a control, 1 mM ATP replaced pyrophosphate in the incubation mixture and ADP was omitted in the assay. The reaction was initiated by addition of coupling enzymes. Additional pyrophosphate-dependent assays were done using 800 ).d of cooled incubation mixture in a l-ml final volume with 0.2 mM NADH and PGA-PK/GAP-DH (5 units each). Reactions were initiated with 5 mM ATP. As a control, 1 IIIM ATP replaced pyrophosphate in the incubation mixture and ATP was omitted in the assay. The reaction was initiated by addition of coupling enzymes. Assay of GTP and UTP-dependent rates. The assay contained, in 1 ml, 0.1 M Trieine, pH 7.8, 8 mM MgCl,, 0.125 mM NADH, 60 mM KCl, 0.2 mM ADP, 5 mM of the indicated nucleotide, 0.02 unit of glycerate kinase, and coupling enzymes: phosphoglycerate mutase, enolase, PK, and LDH (6 units each). The reaction was initiated with 2.05 mM D-glycerate. As a control, 5 mM ATP replaced GTP or UTP in the assays. Product identification Four coupling enzyme systems were used for product determination: PK/ LDH, PGA-PK/GAP-DH, phosphoglycerate mutase/ enolase/PK/LDH, and enolase/PK/LDH. Reactions were monitored at 340 nm until the exhaustion of glycerate. All reaction mixtures contained, in 1 ml, 1 unit glycerate kinase, 1 mM ATP, 2 mM MgCla, 0.25 mM NADH, and 0.164 mM of either D- or Lglycerate. Equilibrium constant of glycerate kinuse. Incubation mixtures contained, in 1 ml, 0.1 M Tricine, pH 7.8, 4 mM MgCla, 0.094 mM ATP, 0.164 mM D-glycerate, 0.295 ITIM ADP, varying concentrations of 3-PGA (0.29-29.0 mM), and 10 ~1 (0.1 unit) of glycerate kinase which was previously dialyzed against 5 mM phosphate buffer, pH 7.4 (degassed). Incubation at 25°C was carried out for 150 min, and then the enzyme was precipitated by freezing at -30°C for 5 h. The change from initial ATP concentration was determined using HK/GGP-DH as coupling enzymes. Assays contained, in 1 ml, 0.1 M Tricine, pH 7.8, 4 mM MgCla, 0.5 mM NAD, aliquots of equilibrium mixture, and 8 units each of coupling enzymes. Reactions were initiated by addition of 10 mM Dglucose, and the reduction of NAD was followed at 340 nm until ATP was exhausted. The equilibrium constant of the glycerate kinase reaction is defined (D-g~yCeK&!). as Keq = (Mg-ATP) (3-PGA)/(Mg-ATP) Initial and equilibrium concentrations of Mg-ATP, Mg-ADP, and 3-PGA in each of the incubation mixtures were calculated as described by O’Sullivan

KINASE

187

KINETICS

and Smithers (18) using the following stability constants (corrected to pH 7.8): KMg.ATP = 69,700 M-l, ~~~~~~~ = 3900 M-l, and Kpda.pC,,= 39 M-i. Kinetic potterns of glycerate kinase. The data points (two to five replicates) were fitted to the appropriate rate equations for sequential Bi-Bi mechanism and for competitive, noncompetitive, and uncompetitive inhibition using a least-square method and computer programs described by Cleland (19). The patterns shown in Figs. 5-9 were drawn for the best fit of computer analysis. Concentrations of free magnesium as well as Mg-ATP were calculated as described by O’Sullivan and Smithers (18), using the following stability constants (corrected to pH 7.8): Khlg.ATP = 69,700 M-I, K,,.pGA = 39 M-i, and KMK:-PEP= 182 M-l. The rates were determined using 0.01-0.03 unit glycerate kinase per 1 ml of assay.

a

b

I

FIG. 1. SDS-polyacrylamide gel electrophoresis of purified glycerate kinase. An aliquot of 5 pg of the enzyme eluted from a Green A column (lane b) and 20 pg standard proteins (lane a) were run under denaturing conditions in a 12.5% polyacrylamide gel. Standard proteins kit (Bio-Rad) contained phosphorylase b (l), i@, 92,500; BSA (2), M, 66,200; ovalbumin (3) M, 45,000; carbonic anhydrase (4), M, 31,000; soybean trypsin inhibitor (5), M, 21,500; and lysozyme (6). M, 14,400. The gel was stained with a silver reagent (Bio-Rad).

188

KLECZKOWSKI,

RANDALL, TABLE

AND

ZAHLER

I

PRODUCTS AND SPECIFICITY OF SPINACH LEAF GLYCERATE KINASE

Substrate

Coupled enzymes

Expected

Relative phosphorylation (%)

product

D-Glycerate L-Glycerate

ADP ADP

D-Glycerate L-Glycerate

D-3-PGA L-3-PGA

P-glyceromutase/ Enolase/PK/LDH

D-Glycerate L-Glycerate

D-&PGA L-3-PGA

Enolase/PK/LDH

D-Glycerate L-Glycerate

D-2-PGA and ADP L-2-PGA and ADP

PK/LDH PGA-PK/GAP-DH

96 6.2

100 6.7

and ADP and ADP

90 6.1 0 0

Note. All assays were done in duplicates.

RESULTS

Purity

AND

DISCUSSION

of the Enzyme

Glycerate kinase from spinach leaves has been purified about 1400-fold to a specific activity of 280 units/mg protein using the procedure of Kleczkowski and Randall (8) with slight modifications. The final enzyme preparation was homogenous as judged by silver staining (Bio-Rad) of SDS-polyacrylamide gels (Fig. 1). The specific activity of the purified enzyme is the highest reported for glycerate kinase from any source. Using the V,,, of the enzyme close to 300 units/mg protein, the calculated turnover number equals 12,000. min-‘.

NADH resulted in another 6-7 or 90100% phosphorylation, respectively. Thus, the enzyme was still fully active at the end of the initial phosphorylation period. These data strongly suggested that Lglycerate was not a substrate but that a small amount of contaminating D-glycerate was present in the L-glycerate stocks.

SpeciJicity Glycerate kinase shows absolute specificity for D-glycerate as the phosphate acceptor (Table I, Fig. 2). The enzyme is not active with either D-gluconate, DLglyceraldehyde, glycerol, glycolate, D-lactate, DL-malate, or DL-serine. While activity was initially observed with L-glycerate, the experiments to exhaustively phosphorylate both D- or L-glycerate indicated that less than 7% of L-glycerate was phosphorylated when compared to the D-stereoisomer (Table I). The addition of second aliquots of either L- or D-glycerate and

\- I '0 I

2I1

4

6I

8I

I / [D-GLYCERATE

IO I

12 I

14 II

I

] ,ImM-'1

FIG. 2. The effect of L-glycerate on glycerate kinase. ATP and MgClz concentrations were fixed at 5 and 10 mM, respectively (LDH/PK system). L-Glycerate was added to the assays (duplicates) immediately before the D stereoisomer. Closed circles and closed squares are points adjusted for 6.7% D-glycerate contamination in 0.82 and 2.95 pmol L-glycerate, respectively.

SPINACH

GLYCERATE

This conclusion was further supported by experiments with both D- and L-glycerate present simultaneously in the assays (Fig. 2). L-Glycerate appears to be a nonlinear “activator”; however, if one calculates substrate concentration assuming a 6.7% contamination of D-glycerate in the Lglycerate stock (Table I), the curves determined in the presence of L-glycerate are superimposable on the curve with the D-stereoisomer alone. The inactivity of glycerate kinase toward L-glycerate and apparent lack of inhibition by this stereoisomer (Fig. 2) suggest that the configuration of the 2-hydroxyl group is critical for binding to the enzyme. The data also indicate that L-glycerate produced by the action of L-lactate dehydrogenase on hydroxypyruvate in lettuce leaves (20) would not be metabolized to L-3-PGA by this glycerate kinase. It seems important to point out that serine does not serve as a substrate for glycerate kinase, indicating that phosphoserine detected in considerable amounts during onset of photosynthesis in leaves (21) must arise by other mechanism(s), most likely from 3-PGA through the coupled reactions of PGA dehydrogenase and phosphoserine aminotransferase (22). While specific for D-glycerate, glycerate kinase does not discriminate between some of phosphate donors. The relative rates with either ATP, UTP, or GTP included into assay mixture were 100, 57, and 42%,

. MrlClp 0

2

4 MgCl2

FIG. 3. The divalent

6 or

8 IO 12 MnCl2 (mM)

14

cation requirement of glycerate kinase. Duplicate assays were done with 2.05 mM Dglycerate in a PK/LDH system.

KINASE

189

KINETICS

-ii 60 a

-401

j

FIG. 4. Determination of an equilibrium constant for glycerate kinase. [P], product of the initial concentration of 3-PGA and Mg-ADP; [S], product of the initial concentration of D-glyCc?rate and Mg-ATP.

respectively. The enzyme was totally inactive toward pyrophosphate as a substrate. With ATP as a preferred donor, the enzyme transfers the y-phosphate only to the 3-hydroxyl group of D-glycerate, yielding ADP and D-3-PGA as the products (Table I). The preferential utilization of ATP as a phosphate donor has been previously shown for glycerate kinase from pea leaves (7). Divalent

Cation Requirement

In the course of experiments, care was taken to maintain an optimal divalent cation concentration for glycerate kinase activity. This requirement is satisfied to the same extent by magnesium or manganese ions (Fig. 3). The enzyme shows an optimal activity with a relatively wide range of magnesium concentration (Fig. 3). At all tested concentrations of ATP, the 3.0 to 5.5 mM excess of total magnesium over total ATP does not affect the rates. Under these conditions essentially all of the ATP is complexed in the form of Mg-ATP, which is the true substrate for glycerate kinase. We kept a similar range of free magnesium during kinetic experiments to assure that all the ATP is complexed and to avoid inhibition by too high levels of the divalent cation. Equilibrium

Constant

The equilibrium constant for the glycerate kinase-catalyzed reaction is 303, as

KLECZKOWSKI,

0

4

0

I2

I I[ D-GLYCERATE]

RANDALL,

16

20

,(mM-‘)

AND

ZAHLER

0

2

4

6

I / [ Mg-ATP]

0

1012

, (mM-‘1

FIG. 5. Substrate kinetics of glyeerate kinase with D-glycerate (A) or ATP (B) as a changing substrate. Assays were done with 5 mM fixed MgClz in PK/LDH system.

shown in Fig. 4. Since this estimate strongly favors the points close to abscissa, we additionally calculated the equilibrium ratios of the reactants for each of the points. The average Keg value calculated by this approach is 283 + 10, which is reasonably close to the value estimated in Fig. 4. Kinetic

at both nonsaturating and near-saturating concentrations of D-glycerate (Figs. 6A, B), while inhibition against D-glycerate yielded noncompetitive patterns in both nonsaturating and near-saturating con-

3-PGA

(mM)

-

Mechanism

Initial velocity experiments with Dglycerate varied at several concentrations of ATP and with ATP varied at several concentrations of D-glycerate yielded linear patterns converging on the abscissa (Figs. 5A, B). The replots of slopes as well as intercepts gave straight lines crossing the ordinate above the origin. These patterns are consistent with a sequential mechanism. K, values taken both from the double-reciprocal plots and from the replots of slopes and intercepts are 0.21 + 0.02 and 0.25 + 0.02 mM for ATP and D-glycerate, respectively. The K, for ATP is somewhat lower than the values reported for rye and pea glycerate kinase [0.655 to 0.692 mM, Ref. (7)], while K, for D-glycerate is similar to those obtained for the enzyme from either species [0.180 to 0.188 mM for both rye and pea enzyme, Ref. (7)]. Product inhibition studies with 3-PGA yielded competitive patterns against ATP

I 0

I I I [~g-ATP]

0

40

2.0

60

,( ~M-‘J

2.0

4.0

60

I /[Mg-ATP].(mM-‘1

FIG. 6. 3-PGA inhibition of glycerate kinase with ATP as a changing substrate at fixed near-saturating (A) or nonsaturating (B) concentrations of D-&X%Xate. Assays were done with fixed concentrations of MgClz (5.4 mM) and D-glyCerate (2.05 and 0.246 mM in A and B, respectively) (PK/LDH system).

SPINACH

3-PGA

r 7’ 3 ‘: P 1

KINASE

191

KINETICS

ATP

(mM)

4 c c5 ‘j B

GLYCERATE

GIY

5 136

?{--+=-g+

2 102

PRODUCTS

ATP

QY 68

E Gly PGA

34

0

t E

0

SCHEME

B lu 2.0

4.0

6.0

8.0

also studied the effect of competitive inhibitors (analogs) of either D-glycerate or ATP on the kinetic patterns obtained with the second substrate. 2-PGA, a competitive inhibitor to ATP (Fig. 8A), yielded noncompetitive patterns with D-glycerate (Fig. 8B), while glycolate, a competitive inhibitor to D-glycerate (Fig. 9A), gave noncompetitive patterns with ATP (Fig. 9B). The replots of slopes and intercepts in Figs. 8 and 9 were linear. These data are consistent only with a random mechanism. Inhibition patterns of glycerate kinase have been summarized in Table II. The proposed sequential random mechanism for glycerate kinase is presented in Scheme 1. From the data presented it is not possible to distinguish between rapid equilibrium and steady-state mechanisms (23). A notable characteristic of the reaction is the formation of a dead-end complex (enzyme-glycerate-PGA), which is indicated by the noncompetitive inhibition by 3-PGA against D-glycerate (Fig. 7). This type of a dead-end complex is quite unusual, as one would expect 3-PGA to be an analog of glycerate. However,

10.0

I / [D-GLYCERATE],

(mM-‘1

3-PGA

(mM 1

9

5 a

7

2.5

5

3

0

2.0

0 4.0

6.0

8.0

I / [D-GLYCERATE]

,hd)

FIG. 7. 3-PGA inhibition of glycerate kinase with D-glycerate as a changing substrate at fixed nearsaturating (A) or nonsaturating (B) concentration of ATP. Assays were done with fixed concentrations of MgCla (10 and 5 mM in A and B, respectively) and ATP (5 and 0.2 mM in A and B, respectively) (PK/ LDH system).

centrations of ATP (Figs. 7A, B). The replots of slopes and intercepts in Figs. 6 and ‘7 gave straight lines with the exception of Fig. 7B, where the replot of slopes was nonlinear. As these data were insufficient to distinguish between ordered and random mechanism for the enzyme, we

“0 vz ” -.G a, ‘0 h E” E” 7 3 --Z P 1

260 26OtA

2-PGAhM)

1.

1 -

20 F; al ‘0 h

195 IO 130

0 5

65

0

,

,

2.0

4.0

I I/ [~g-ATP] [Mg-ATP]

,lZo

.(mM-‘) .c~M?

6.0

(,,,,, 0

1 2.0

4.0

11 I/ [D-glycerate]

6.0

8.0

10.0

,(mM-‘)

FIG. 8. 2-PGA inhibition of glycerate kinase with ATP (A) or D-glycerate (B) as a changing substrate. Assays were done with fixed concentrations of MgClp (5.4 mM) and D-glycerate (0.246 mM, A) or ATP (0.2 mM, B) (PK/LDH system).

192

KLECZKOWSKI,

RANDALL,

AND

“o -

GLYCOLATE

27-8

20

40

6.0

8.0

11 [D-GLYCERATE]

(mM 1

-

34

So 1 0

ZAHLER

I 2.0

0

10.0

,(~M“I

I / [Mg-ATP]

4.0

6.0

, CrnM-‘)

FIG. 9. Glycolate inhibition of glycerate kinase with D-glycerate (A) or ATP (B) as a changing substrate. Assays were done with fixed concentrations of MgCla (5.2 mM and 5.4 mM in A and B, respectively) and ATP (0.2 mM, A) or D-giycerate (0.271 mM, B) (PK/LDH system).

inhibition by 3-PGA vs ATP was determined at two concentrations of the second substrate and is clearly competitive (Fig. 6). Apparently, the phosphate group is a key determinant in binding. Our data do not exclude the possibility of additional dead-end complex(es)-especially enzymeglycerate-ADP, which would be expected for a kinase having a random mechanism (24). Regulation

Table III presents the effect of various metabolites on the activity of glycerate TABLE SUMMARY

Fixed substrate ATP (0.20 mM) ATP (5.0 mM) D-Giycerate (0.246 mM) D-Giycerate (2.05 mM) D-Giycerate (0.246 mM) ATP (0.20 mM) ATP (0.20 mM) D-Glycerate

(0.271IIIM) a Noncompetitive. * Competitive.

kinase at concentrations of D-glycerate and ATP close to their Km values. Phosphorylated metabolites are seen to be better inhibitors than the nonphosphorylated compounds, suggesting a role for phosphate in inhibition. Since phosphorylated compounds can frequently chelate WC?+, one possibility is that apparent inhibition results from competition for free Mg2+. However, calculations show that 8 mM pyrophosphate, the strongest chelator used (18), will cause only a 2% decrease in Mg-ATP under conditions of the assays. Thus, while chelation may II OF SPINACH LEAF GLYCERATE

KINASE

OF THE INHIBITION

PATTERNS

Changing substrate

Inhibitor

&

D-Glycerate D-Glycerate

3-PGA 3-PGA

0.72 * 0.12 9.3 + 3.2

ATP

3-PGA

0.60 r 0.06

C*

ATP

3-PGA

1.5 f 0.1

C

ATP D-Giycerate

2-PGA 2-PGA

D-Glycerate

Glycolate

11.7 f 1.0 19.1 f 1.5 16.0 f 2.1

42.3 f 2.9 -

C NC C

ATP

Glycolate

22.3 f 6.4

46.2 + 9.5

NC

(mM)

K,, (mM) 2.3 f 0.6 9.9 * 2.0

Pattern NC” NC

SPINACH

GLYCERATE

contribute slightly to the inhibition at high pyrophosphate concentration, it is not a significant factor in the overall results. The competitive inhibition patterns against ATP shown with 3-PGA and 2-PGA (Figs. 6, 8), as well as with phosphoglycolate and pyrophosphate (data not shown), strongly suggest that the presence of a phosphate group directs binding of these compounds to the ATP site on the enzyme. The effect of phosphorylated

TABLE

III

METABOLIC COMPOUNDS AS INHIBITORS OF SPINACH LEAF GLYCERATE KINASE

Compound DL-Alanine (20 mM) D-Gluconate (20 mM) DL-Glyceraldehyde (20 mM) L-Glycerate” (2.95 mM) Glycine (20 mM) Glycerol (20 mM) Glycolate (20 mM) Hydroxypyruvate* (10 mM) D-Lactate (10 mM) D-Lactate* (10 mM) D-Malate (20 mM) DL-Serine (20 mM) Carbamyl phosphate (8 mM) DHAP (8 mM) Fructose 1,6-bisphosphate (8 mM) Fructose 2,6-bisphosphate (8 PM) D-Glucose 6-phosphate (8 mM) D-2-PGA (8 mM) D-2,3-PGA (8 mM) D-3-PGA (8 mM) 6-Phosphogluconate (8 mM) Phosphoglycolate (8 mM) Phosphate (8 mM) Pyrophosphate (1.5 mM) Pyrophosphate (8 mM) o-Ribose 5-phosphate (8 mM) D-Ribulose 1,5-bisphosphate (8 mM)

Relative activity (%I 90 82 80 100 92 100 63 63 54 59 56 96 87 87 66 102 94 68 63 27 56 61 80 69 11 84 39

Note. All assays (duplicates) contained 0.2 mM ATP, 0.246 mM D-glycerate, and 5.2 mM MgCl,. All compounds except those marked with (b) were assayed in PK/LDH system. ’ See Fig. 2. b Assayed in PGA-PK/GAP-DH system.

KINASE

KINETICS

193

compounds might offer a simple regulation scheme for glycerate kinase in vivo with the photosynthetic intermediates (e.g., 3PGA, RuBP) acting as negative effecters during the light conditions. However, there are several factors which point against this scheme, the major ones being the relatively high affinity of the enzyme for ATP (K, = 0.21 mM, Fig. 5B) and the near-saturating concentration of ATP reported for stromal compartments during light conditions (25, 26). It is also known that, upon illumination, there is an increase in the stromal level of D-glycerate [from 0.46 to 1.19 mM, Ref. (4)], which would consequently increase the rates of glycerate kinase. Our results are in agreement with the proposal of Schmitt and Edwards (7) that glycerate kinase activity is modulated solely by substrate availability functioning as a linkage between photorespiration and reductive photosynthetic carbon pathway. REFERENCES 1. TOLBERT, N. E. (1971) Annu. Rev. Plant Physiol 22.45-74. 2. TOLBERT, N. E. (1981) Annu. Rev. B&hem. 50, 133-157. 3. HEBER, U., KIRK, M. R., GIMMLER, H., AND SCHAFER, G. (1974) Plunta 120, 31-46. 4. ROBINSON, S. P. (1982) B&hem. Biophys. Res. Commun. 106, 1027-1034. 5. HATCH, M. D., AND SLACK, C. R. (1969) B&hem. Biophys. Res. Commun 34, 589-593. 6. USUDA, H., AND EDWARDS, G. E. (1980) Plant PhysioL 65, 1017-1022. 7. SCHMITT, M. W., AND EDWARDS, G. E. (1983) Arch. Biochem Biophys. 244, 332-341. 8. KLECZKOWSKI, L. A., AND RANDALL, D. D. (1983) FEBS l&t. 158, 313-316. 9. BLACK, S., AND WRIGHT, N. G. (1956) J BioL Chem. 221, 171-180. 10. OZAKI, K., AND WETTER, L. R. (1960) Cmad J. B&hem PhysioL 38, 125-131. 11. ICHIHARA, A., AND GREENBERG, D. M. (1957) J. BioL Chem. 225, 949-958. 12. KATAYAMA, H., KITAGAWA, Y., AND SUGIMOTO, E. (1980) J. B&hem. 88, 765-773. 13. LAMPRECHT, W., DIAMANTSTEIN, T., HEINZ, F., AND BALDE, P. (1959) Z. PhysioL Chem. 316, 97-112.

194

KLECZKOWSKI,

RANDALL,

14. HANSEN, R. W., AND HAYASHI, J. A. (1962) J. Bacterial. 83, 679-687. 15. DOUGHTY, C. C., HAYASHI, J. A., AND GUENTHER, H. L. (1966) J. Biol. Chem. 241, 568-572. 16. ORNSTON, M. K., AND ORNSTON, L. N. (1969) J. Bactwiol. 97, 1227-1233. 17. HILL, B., AND ATTWOOD, M. M. (1974) J. Gen Microl?iol 83, 187-190. 18. O’SULLIVAN, W. J., AND SMITHERS, G. W. (1979) in Methods in Enzymology (Purich, D. L., ed.), Vol. 63, pp. 294-336, Academic Press, New York. 19. CLELAND, W. W. (1979) in Methods in Enzymology (Purich, D. L., ed.), Vol. 63, pp. 103-138, Academic Press, New York.

AND

ZAHLER

20. BETSCHE, T. (1981) Biochem. J. 195, 615-622. 21. DALEY, L. S., AND BIDWELL, R. G. S. (1977) Plant Physiol. 60, 109-114. 22. LARSSON, C., AND ALBERTSSON, E. (1979) Physiol. Plant 45,7-10. 23. SEGEL, I. H. (1975) Enzyme Kinetics, pp. 646649, Wiley-Interscience, New York. 24. CLELAND, W. W. (1970) in The Enzymes (Boyer, P. D., ed.), Vol. 2, pp. 1-61, Academic Press, New York. 25. HEBER, U., TAKAHAMA, U., NEIMANIS, S., AND SHIMIZU-TAKAHAMA, M. (1982) Biochim. BZ(F phys. Acta 679, 287-299. 26. SANTARIUS, K. A., AND HEBER, U. (1965) B&him. Biophys. Acta 102, 39-54.