Archives of

Hicrobiology

Arch. Microbiol. 107, 189-192 (1976)

9 by Springer-Verlag 1976

Unidirectional Inhibition of Phosphoenolpyruvate Carboxykinase from Rhodospirillum rubrum by ATP JOBST-HEINRICH KLEMME Institut ffir Mikrobiologie der Universitfit, Meckenheimer Allee 168, D-5300 Bonn 1,

Federal Republic of Germany

Abstract. The kinetic and regulatory properties o f partially purified p h o s p h o e n o l p y r u v a t e (PEP) carboxykinase (EC 4.1.1.32) f r o m Rhodospirillum rubrum were studied. The enzyme was active with guanosineand inosinephosphates and must thus be classified as G T P (ITP) : oxaloacetate carboxylyase (transphosphorylating). In the direction o f oxaloacetate-formation, the enzyme was strongly inhibited by A T P (Ki = 0.03 m M ) . ITP, U T P , C T P and G T P were less inhibitory. The inhibition was competitive with respect to G D P or I D P , but not with respect to PEP. In the direction o f PEP-synthesis, the enzyme was not inhibited, but rather activated by A T P .

Key words." P E P - c a r b o x y k i n a s e rubrum - Unidirectional inhibition.

Rhodospirillum

P h o s p h o e n o l p y r u v a t e (PEP) carboxykinase (EC 4.1.1.32) catalyzes the reversible nucleoside diphosphate-dependent carboxylation o f P E P to oxaloacetate. The enzyme is considered to be responsible for gluconeogenetic P E P - f o r m a t i o n f r o m C4-dicarboxylic acids in m a m m a l i a n tissues and yeast and was also d e m o n s t r a t e d in the two p h o t o t r o p h i c bacteria Rhodospirillurn rubrum (Evans, 1965; C o o p e r and Benedict, 1968) and Rhodopseudomonas sphaeroides ( U c h i d a and Kikuchi, /966). A l t h o u g h the enzyme f r o m R. rubrurn was obtained in a fairly pure state ( C o o p e r and Benedict, 1968), n o t h i n g was reported concerning a possible regulation o f its activity. C o n sidering the reversibility o f the enzyme reaction and the occurrence o f two PEP-synthesizing enzymes (PEP-carboxykinase and " P E P - s y n t h e t a s e " ) in R. rubrurn ( C o o p e r and Benedict, 1968; Buchanan, 1974), a study o f the kinetic properties o f P E P - c a r b o x y k i n a s e o f R. rubrum seemed o f great interest. It will be shown in this c o m m u n i c a t i o n that A T P is likely to be the

effector which ensures the p r o p e r functioning o f the enzyme in the direction o f P E P - f o r m a t i o n . MATERIAL AND METHODS Rhodospirillum rubrum strain $1 was obtained from Dr. H. Gest, Department of Microbiology, Indiana University, Bloomington, and was grown in liquid cuitures with DL-malate and (NH4)2SO4 (Ormerod et al., 1961). In some experiments, malate was replaced by another carbon source. The cultures were grown at 30~ and about 3000 lux in screw cap-bottles or, under N2, in the 10 1fermentor "Kiel" of L. Eschweiler, Kiel (pH-control set to 6.8), and were harvested when the turbidity at 660 nm was 1.5, corresponding to 750 mg dry weight/1. Enzyme activities are expressed as units. 1 unit is the activity catalyzing the formation of 1 gmole product per min at 30~C. PEP-carboxykinase activity in the direction of oxaloacetateformation was measured spectrophotometrically at 30~C in malatedehydrogenase-coupled reaction mixtures as described in a previous paper (Klemme, 1973). The reaction was started by the addition of enzyme and was linear for at least 2 min. Initial rates were proportional to protein concentrations (determined by the biuret method) in the range from 0-60 gg for the partially purified enzyme. Activity in the direction of PEP-formation was assayed at 30~ by a colorimetric method based on the quantitative conversion of the phosphoryl group of PEP to inorganic phosphate (Pi) in the presence of Hg2+ as described by Cannata and DeFlombaum (/974). Pi in the samples was measured according to the method of Taussky and Shorr (1953). PEP-carboxylase (EC 4.1.1.37) was measured in the malate-dehydrogenase-coupled reaction system described by Canovas and Kornberg (1969). The activity of the PEP-synthetasewas assayed cotorimetrically by following the ATP-dependent disappearance of pyruvate from Pi-containing reaction mixtures (Cooper and Kornberg, 1965).

RESULTS Cells g r o w n photosynthetically with different c a r b o n sources (acetate, lactate, malate, succinate, fructose) were analyzed for P E P - c a r b o x y k i n a s e titers. C o n t r a r y to the enzyme f r o m baker's yeast (Haarasilta and Oura, 1975), the Rhodospirillum rubrum P E P - c a r b o x y k i n a s e is constitutive with activity levels ranging f r o m 0 . 1 0.2 units/mg protein.

Arch. Microbiol.,Vol. 107, No. 2 (1976)

190 Table 1. Partialpurification of PEP-carboxykinasefrom Rhodospirillum rubrum Fraction

140000 g-supernatant Protamine sulfate supernatant (NH4)zSO4-precipitate(50- 70 ~osat.) Heat step (55~C, 5 min) supernatant Sephadex G-200 filtrate a u

ml

60 65 12 10 30

Protein (mg)

1200 960 220 98 23

PEP-carboxykinase Total units

Spec. activity (units/rag protein)

132" 120~ 80~ 72a 49~

0.11 ~ 0.12~ 0.368 0.73a 2.10a

0.4g b

1.05b 2.92b

Assayin the direction of oxaloacetate-formation(GDP). Assayin the direction of PEP-formation (ITP).

The enzyme was partially purified from cells grown photosynthetically with malate by using conventional techniques such as treatment of the ultrasonic extract with protamine sulfate (0.1 mg/mg protein), (NH4)2SO4-fractionation, heat denaturation and gelfiltration. The enzyme was stabilized by addition of 2 m M reduced glutathione, 1 m M MnSO4 and 1 m M ATP to the basal phosphate buffer (10 raM, pH 7.5). The elution position of the enzyme (V~/Vo = 1.7) from a Sephadex G-200-column corresponded to a molecular weight of about 80000. The purification procedure resulting in a 20-fold increase of specific activity is summarized in Table 1. The enzyme preparation was free of pyruvate kinase, "PEP-synthetase" and PEP-carboxylase, but still contained a considerable malate dehydrogenase-activity. Consequently, the activity of the PEP-carboxykinase in direction of PEP-formation could not be measured with a N A D H coupled assay system. As shown in Table 1, the specific activity of the enzyme was about the same in both directions. With the partially purified enzyme, the kinetics of oxaloacetate-formation were studied. With saturating conditions (5 m M PEP, 0.5 m M GDP, 25 m M NaHCO3, 1 m M MnSO4), the pH-optimum of the reaction was at p H 7.5. The enzyme showed a low degree of specificity to nucleoside phosphates. G D P (100), IDP (46), CDP (23) and U D P (18) served as substrates, the relative activities obtained with 0.5 m M nucleoside diphosphate being given in the brackets. With ADP, the enzyme was practically inactive. With G D P and IDP as variable substrates, normal hyperbolic saturation curves with Kin-values of 0.013 (GDP) and 0.12 ( I D P ) m M were obtained. However, with PEP as the variable substrate, the enzyme exhibited a biphasic saturation curve with a transition point at about 0.4 m M PEP and half-saturation at about 1.4 m M PEP. A variety of metabolites were tested for possible effects on the enzyme. P~, C4-dicarboxylic acids and

sugar phosphates were without effect. However, ATP was a potent inhibitor of the enzyme. U T P (88), CTP (88), ITP (80) and G T P (53) were less effective, the relative inhibitory capacities with respect to ATP (100) being given in the brackets. The possibility that the inhibition by ATP was due to chelation of Mn 2 + could be excluded on the ground of the high affinity of the enzyme to Mn 2 + (So.s = 80 I~M). The extent of inhibition by ATP was dependent on the GDPconcentration but independent of the PEP-concentration. When initial reaction rates were measured as a function of ATP-concentration at various, fixed GDP-concentrations, the reaction data did not give straight lines in the Dixon-plot (1/v versus inhibitor concentration) with 0.2 and 0.5 m M G D P (Fig. 1). However, when the linear portions of the inhibition curves were extrapolated, a common point of intersection was obtained indicating the competitive nature of the inhibition and allowing an estimation of the Ki for ATP (0.03 raM). Another experiment showed that ATP also interfered with the binding of IDP to the enzyme. High concentrations of IDP (3 mM) released the inhibition by 1 m M ATP. The kinetics of PEP-formation were studied at the optimal p H of 9.0. In a reaction mixture with 2 m M oxaloacetate, 2 m M ITP and 1 m M MnSO4, the reaction rate remained constant for only about 3 min (Fig. 2). Using G T P instead of ITP as a phosphate donor, the reaction was linear for only 1 min and the reaction rate was 50~o lower. ATP was practically inactive as a phosphate donor. In reaction mixtures with i m M ATP and an active nucleoside triphosphate (ITP or GTP), the initial reaction rates were not significantly changed. However, the rates remained constant for at least 5 min (Fig.2). It is concluded that the apparent "activation" of the reaction by ATP can be attributed to the inhibition of G D P - or IDP-binding to the enzyme. Due to this inhibition, the approach of the kinetic equilibrium between forward and back reaction would be delayed.

J.-H. Klemme: ATP-Inhibition of R. rubrum PEP-Carboxykinase

3c

8C

-/

20

6C >.

>_-

4C 2(

o.1r~M.-'~ ~ GDP ~

~0,

o.'5

,io

[ATP]

7,(~o

O.S ~.0 [ATP]{mM)

' //~'.o

(mM)

Fig. J. ATP-inhibition of Rhodospirillum rubrum PEP-carboxykinase assayed in the direction of oxaloacetate-formation. The reaction mixture contained 100 mM Tris-HC1 (pH 7.5), 25 mM NaHCO3, I mM MnSO4, 5 mM PEP, 55 pg of partially purified enzyme and the indicated amounts of GDP and ATP. 100K activity corresponds to a AAaa4/min of 0.155, 0.I85 and 0.190 for 0.1 (O), 0.2 (A) and 0.5 (9 mM GDP, respectively. The inset shows the Dixon-plot of the reaction data

ITP

1.0 o ZL 0.5 o

~

,

o

CoTp

,

5

10

15

rnin

Fig. 2. Kinetics of PEP-formation catalyzed by Rhodospirillum rubrum PEP-carboxykinase. The reaction mixture contained 100 mM Tris-HCI (pH 9.0), 1 mM MnSO4, 2 mM oxaloacetic acid, l l 0 p g of partially purified enzyme and various nucleoside triphosphates: 2 m M ATP (A); 2 r a m GTP (O); 2raM ITP (e); 2mM GTP + 1 mM ATP (A); 2 mM ITP + 1 mM ATP (F1)

The Kin-values for ITP and GTP in the presence of 2 mM oxaloacetate and 1 mM ATP were found to be lower than 0.05 raM. Thus, for a more precise estimation, an enzyme preparation free of malate dehydrogenase-activity has to be available allowing the use of the NADH-coupled spectrophotometric assay.

DISCUSSION Unlike Rhodopseudomonas sphaeroides in which the combined action of pyruvate carboxylase and PEPcarboxykinase is responsible for gluconeogenetic PEPformation (Uchida and Kikuchi, 1966; Payne and Morris, J969), Rhodospirillum rubrum contains a Pi-

191

dependent enzyme catalyzing PEP-formation from pyruvate and ATP (Buchanan and Evans, 1966; Buchanan, 1974). Considering now the possibility of PEP-synthesis from C4-dicarboxylic acids by the combined action of malic enzyme (EC l.i.J.38) and PEP-synthetase (Hansen and Juni, 1974), it may be asked in which direction the PEP-carboxykinase is functioning in R. rubrurn [besides "PEP-synthetase", R. rubrurn contains malic enzyme (Liideritz and Klemme, unpublished)]. There are two ways by which a reversible enzyme reaction can be made '"unidirectional" in vivo. One such mechanism concerns the steady state concentrations of substrate and product. As an example, the pyrophosphorolytic group of enzymes shall be mentioned. The function of these enzymes is made unidirectional by a nearly complete removal of inorganic pyrophosphate by the potent enzyme PPase. In the second case, enzyme function is made unidirectional by allosteric ligands which are effective only in one direction of catalysis. As an example, the glutamate dehydrogenase of various Oomycetes may be quoted. This enzyme is activated by NADH, NADPH and PEP. The activators operate unidirectionally in that it is the reductive amination which is effectively controlled (LeJohn and Stevenson, 1970). It is concluded that the action of PEPcarboxykinase in R. rubrum is made unidirectional by the efficient ATP-inhibition of GDP- or IDP-binding to the enzyme. Under the conditions prevailing in the growing bacterial cell (ATP-pool of about 3 mM, PEP-pool of probably not more than 1 raM, nucleoside diphosphate-pool of probably not more than i raM), oxaloacetate-formation catalyzed by PEP-carboxykinase would be nearly completely blocked. Thus, it is very likely that during growth on C4-dicarboxylic acids, the enzyme PEP-carboxykinase functions as the PEP-regenerating enzyme in R. rubrum. Acknowledgements. The author wishes to thank Horst K6cher for skilful technical assistance. The work was supported by grants from the Deutsche Forschungsgemeinschaft.

REFERENCES Buchanan, B. B.: Orthophosphate requirement for the formation of phosphoenolpyruvate from pyruvate by enzyme preparations from photosynthetic bacteria. J. Bact. 119, 1066-1068 (1974) Buchanan, B. B., Evans, M. C. W. : The synthesis of phosphoenolpyruvate from pyruvate and ATP by extracts of photosynthetic bacteria. Biochem. biophys. Res. Commun. 22, 484--487 (i 966) Cannata, J. J. B., DeFlombaum, M. A. C.: Phosphoenolpyruvate carboxykinase from bakers' yeast. Kinetics of phosphoenolpyruvate formation. J. biol. Chem. 249, 3356-3365 (1974) Canovas, J. L., Kornberg, H. L. : Phosphoenolpyruvate carboxylase from Escherichia coli. In: Methods in enzymology, Vol. 13 (S. P. Colowick, N. O. Kaplan, eds.), pp. 288-292. New York: Academic Press J969

192 Cooper, T. G., Benedict, C. R. : PEP carboxykinase exchange reaction in photosynthetic bacteria. Plant Physiol. 43, 788-792 (1968) Cooper, R. A., Kornberg, H. L.: Net formation of phosphoenolpyruvate from pyruvate by Escherichia coli. Biochim. biophys. Acta (Amst.) 104, 618-620 (1965) Evans, M. C. W.: The photoassimilation of succinate to hexose by RhodospirilIum rubrum. Biochem. J. 95, 669-677 (1965) Haarasilta, S., Oura, E. : On the activity and regulation of anaplerotic and gluconeogenetic enzymes during the growth process of baker's yeast. The biphasic growth. Europ. J. Biochem. 52, 1 - 7 (1975) Hansen, E. J., Juni, E. : Two routes for synthesis of phosphoenolpyruvate from C4-dicarboxylic acids in Escherichia coli. Biochem. biophys. Res. Commun. 59, 1204-1210 (1974) Klemme, J.-I-I.: Allosterische Kontrolle der Pyruvatkinase aus Rhodospirillum rubrum durch anorganisches Phosphat und Zuckerphosphatester. Arch. Mikrobiol. 90, 305-322 (1973)

Arch. Microbiol., Vol. 107, No. 2 (1976) LeJohn, H. B., Stevenson, R. M.: Multiple regulatory processes in nicotinamide adenine dinucleotide-specific glutamic dehydrogenase. J. biol. Chem. 245, 3890-3900 (1970) Ormerod, J. G., Ormerod, K. S., Gest, H.: Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch. Biochem. Biophys. 94, 449-463 (1961) Payne, J., Morris, J2 G.: Pyruvate carboxylase in Rhodopseudomonas spheroides. J. gen. Microbiol. 59, 97-101 (1969) Taussky, H. H., Shorr, E.: A microcolorimetric method for the determination of inorganic phosphorus. J. biol. Chem. 202, 675-685 (1953) Uchida, K., Kikuchi, G.: Phosphoenolpyruvate carboxykinase from Rhodopseudomonas spheroides and its possible role in light-stimulation of glucogenesis. J. Biochem. 60, 729-732 (1966) Received October 1, 1975