Eur. J. Biochem. 59,347-353 (1975)
The Pyruvate-Dehydrogenase Complex from Azotobacter vinelandii 2. Regulation of the Activity Tjarda W. BRESTERS, Arie DE KOK, and Cees VEEGER Department of Biochemistry, Agricultural University, Wageningen (Received June 25/August 27, 1975)
The presence of activators (AMP and sulphate) or inhibitors (acetyl-CoA) has no influence on the Hill coefficient of the S-shaped [pyruvate] - velocity curve of either the pyruvate-NAD' overall reaction ( h = 2.5) or that of the pyruvate-K,Fe(CN), activity of the first enzyme (11 = 1.3). pH studies indicated that the Hill coefficient is dependent on subunit ionization within the pyruvate-containing complex and not on those in the free complex. It is concluded that pyruvate conversion rather than pyruvate binding is responsible for the allosteric pattern. The activity is, due to absence of a protein kinase, mainly regulated at the acetyl-CoA/ CoA, and NADH/NAD+ levels and by the value of the energy charge.
Differences have been observed between the mechanisms of regulation of the activity of the pyruvate dehydrogenase complexes from mammals and bacteria. The activity is inhibited in both cases by the products of pyruvate oxidation : acetyl-CoA and NADH [l - 41. The inhibition by acetyl-CoA is reversed by CoA in the mammalian system and by pyruvate in the Eschericltia coli system. NADH inhibition is exerted through action of lipoamide dehydrogenase and is competitive with respect to NAD'. The relative importance of product inhibition for the mammalian system remains questionable, since Linn and co-workers [5,6] and Wieland and von Jagow-Westermann [7] described the regulation by a phosphorylation and dephosphorylation process. Phosphorylation and concomitant inactivation is catalyzed by an ATP-specific kinase. The activity is restored by the action of a phosphatase. Kinase and phosphatase are both M&+-dependent. By combined action the activity of the complex can be regulated by the ATP/ADP ratio. Abbreviation. TPP, thiamine pyrophosphate. Enzymes. Pyruvate dehydrogenase complex is pyruvate-NAD'
reductase, decarboxylating, CoA acetylating (EC 1.2.4.1); pyruvate dehydrogenase is pyruvate-K,Fe(CN), reductase, decarboxylating (EC 1.2.2.2); transacetylase or lipoate acetyltransferase (EC 2.3.1.12); lipoamide dehydrogenase (EC 1.6.4.3): phosphotransacetylase or phosphate acetyltransferase (EC 2.3.1.8); citrate synthase (EC 4.1.3.7); carnitine acetyltransferase (EC 2.3.1.7); myokinase or adenylate kinase (EC 2.7.4.3).
Such a type of regulation does not exist in E. coli [8]. Shen et ul. [9,10] proposed that the complex of E.coli is subject to regulation by the energy charge and also to the concentration of negative feedback effectors. The enzyme pyruvate dehydrogenase which catalyzes the first step of the overall process seems to be the main site for the regulation by energy charge. In this paper the regulation of the Azotobucter vinelandii complex is described and compared with the properties of complexes from other sources.
MATERIALS AND METHODS Materials and the methods for the determination of the activity of the partial reactions of the enzymes of the complex as well as for the overall reaction are given in the preceding paper [ll]. The pyruvate dehydrogenase complex used in these studies was purified up to step4 and had a specific activity of 7 units . mg protein-'. It contained some phosphotransacetylase. The CoA concentration was calculated from - SH group measurements according to Ellman [12] and the acetyl-CoA concentration with a system containing oxaloacetate, citrate synthase and 5,5'-dithiobis(2nitrobenzoic acid) ( E = 13600 M-' cm-l at 412 nm, pH 8.0). The CoA-regenerating systems used consisted either of 0.2 mM oxaloacetate plus 3.5 enzyme units
348
Regulation of Pyruvate-Dehydrogenase Complex from A . vinelundii
of citrate synthase or 20 mM carnitine plus 2 enzyme units of carnitine acetyltransferase. Desired values of the energy charge of the adenylate pool were obtained with the aid of myokinase after mixing the proper concentrations of AMP, ADP and ATP [13]. An equilibrium constant of 2.26 (10 mM MgCl,, pH 7.4, 25°C) was used for the reaction 2 ADP eATP AMP according to Bergmeyer [ 141.
/
+
RESULTS AND DISCUSSION Efiect of M g 2 + . T P P on the Activity
The activity of purified preparations from different sources are partially dependent on M$+ or Ca2+ as cofactor [15- 171, whereas these metals are not required for the activity of the complex from Neurospora c r a m [ 181. Purified pyruvate dehydrogenase complex from A . vinelandii contains small amounts of Mg2+ as judged from the low residual activity, when no metal was added to the reaction mixture; EDTA inhibits this activity. Frequently MnZ+ can be used in M$+dependent reactions. From the influence of the metal concentration on the overall activity, measured in 0.05 M Tris-HC1 (pH 7.6) under standard conditions, the K , values for MgZf and Mn2+ were found to be 100 pM and 10 pM respectively (Fig. 1). CaZ+acts as a competitive inhibitor with respect to Mg2+ ( K i = lOpM) and does not induce activity in the absence of M$+. A K, = 25 pM was obtained for the Mgzf .TPP complex varying the concentration of TPP in the presence of 10 mM MgZ (cJ [9]). At higher pyruvate concentrations (10 mM), the K,,, for M$+ . TPP decreases to 10 pM. Thus it is clear that the K, for Mgz+ as determined from Fig. 1 does not reflect the affinity of the enzyme for Mg". Under the conditions of this experiment the K , for M$+ .TPP is 50 pM, in good agreement with the values found at high Mg" concentrations. F i g 1 also shows that the dependence of thc activity on the Mg2+-TPP concentration is greatly influenced by the presence of AMP and the reaction product acetyl-CoA. Sulphate, and to a less extent phosphate, enhance like AMP the affinity of the enzyme for M$+ . TPP. Acetyl-CoA decreases the affinity and behaves as a competitive inhibitor (Ki= 10 pM) with respect to pyruvate (c$ [ll]).
50 1/ [ Mg2'
0
100
. TPP] (rnM-')
Fig. 1. Effect of M$ and Mg2 . T P P on the overall octivity of' the pyruvate dehydrogenase complex of' A. vinelandii in the absence or presence of' ucetyl-CoA and A M P respectively. Reaction mixture as described [ l l ] in the presence of 5 mM pyruvate, 0.5 mM TPP and 0.05 M Tris-HCI. The final pH was 7.6. The M$+ concentrations arc indicated in the figure. The concentrations Mg2+. TPP are calculated using a Kd of 0.41 mM for the M$+ . TPP complex [19].Tempcrature:25"C.(x-x)Noeffectorpresenl;(AA) in the presence of 0.1 mM AMP; (-0) in the presence of 1 niM in thc presence of 10 pM acetyl-CoA AMP; (-0) +
+
+
Dependence of the Activity on the Pyruvate Concentration
Fig.2 shows the rate of the overall reaction at different pyruvate concentrations. Pyruvate exerts a positive homotropic effect. AMP and sulphate stimulate the activity at lower pyruvate concentration. Acetyl-CoA inhibits competitively; this inhibition can
0
0
1
2
3
4
5
6
7
8
9
10
co
[Pyruvate] (mM)
Fig.2. Depeiidence of the rate of thr ovc+-all reac,iiorr on the pyruvate concentrution in the presence or abstwcr ~flecrors. Reaction mixture as described [ l l ] in the presence of 5 mM Mg2+. 0.5 mM TPP and 0.05 M Tris-HCI, final pH 7.5, at 27°C. The production of NADH was measured on the Aminco-Chance dualwavelength spectrophotometer (380- 345 nm) using the 5-20 ?,; A ) in the prestransmission scale. (U No) additions: (A in thc presence of 0.1 mM ence of 10 pM acetyl-CoA; (-) AMP; ( x ~x ) in the presence of 0.1 mM sulphate
be reversed by the positive effectors. It is convenicnt in allosteric proteins to use the substrate concentration at half saturation ( S o , s ) ,since K , cannot be calculated from curves which dcviate from MichaelisMenten kinetics [20]. So.5 = 1.9 mM for pyruvate under the conditions used. In Fig. 3 a Hill plot of the same results is presented. Maximum velocities were extrapolated from the
349
T. W. Bresters. A. de K o k , and C. Veeger
Table 1. Influence ojrhe eflector concentration on the overall reaction of the pyruvate dehydrogenase complex from A. vinelandii Eo,5, the concentration of effector required for half maximal effect, was determined from a curve of reaction rate as a function of the effector concentration at the pyruvate and Mg2+ . TPP concentrations indicated. All curves were hyperbolic Effector
Type of action'
+ +
AMP ADP
+
Acetyl-CoA
-
so:-
Pyruvate
M2'
2 2 0.4 0.16
0.5
60
0.5
160 140
.TPP
0.4 0.4
8
+ is stimulation, - is inhibition.
Fig. 3. Hill plots of the data pwsented in Fig. 2. The same symbols wcre uscd; details, see text
l/velocity V E ~ S U Sl/[pyruvate] curves. The Hill coefficients ( h ) as calculated from the plots are in all cases 2.6-2.7, thus it must be concluded that the cooperativity is not influenced by the presence of the effectors. The shapes of the curves are not dependent on the protein concentration. The effects as observed with AMP and sulphate in stimulating the activity and reversing the acetylCoA inhibition at low pyruvate concentrations are also exerted with decreasing efficiences by ADP, GMP, GDP, phosphate, arsenate, maleate, fumarate and malonate. ATP is slightly inhibitory at 5 mM concentration. The stimulation by AMP or sulphate is not dependent on the presence of sulfhydryl compounds like dithiothreitol in the reaction mixture. In the absence, however, I/ is about 202, lower either with or without the effector (cJ[lo]). In Table 1 the influence of some of the strongest effectors is summarized. In using the alternative electron acceptor ferricyanide to locate the influence of the effectors on the first enzyme of the complex, the effects of AMP and acetyl-CoA are still present and compete with each other or with pyruvate. Flow of electrons to ferricyanide via the lipoyl moieties of the second enzyme will not occur as can be concluded from experiments in which the lipoyl moieties are blocked with N-ethylmaleimide [21]. Fig. 4 shows that the Hill coefficients are 1.3(k0.2). At least part of the characteristics seem to be present
-1.6
-0.8
0
0.8
log [Sl (mM)
Fig. 4. Hill plots of the ferric.).anide-lirihe(~pyruvate deliydrogmuse uctiviry. The activity was measured in Tris-HCI as described [I 11 with the whole complex. The pyruvate concentrations were as indicated. 1' was obtained from Lincwcavcr-Burk plots. ( A ----A) No effector present; ( x -x ) in the presence of 10 pM acetylCoA; (M) in the presence of 20 pM acetyl-CoA; (M) in the presence of 1 mM AMP
in the ferricyanide-linked pyruvate dehydrogenase (partial) reaction. On the other hand one could argue whether, although consistent, such a deviation from h = 1.O is significant. Especially in view of the observation that in the pyruvate oxidase reaction. on which AMP and acetyl-CoA also exert their typical effects, no cooperative effects by pyruvate are observed
(4[111). The Influence of the p H the Rate of the Overall Reaction
OII
The influence of the pH on the velocity was studied with 1 mM NAD' and varying pyruvate concentrations at several pH values. Before and after the reac-
350
Regulation of Pyruvate-Dehydrogenase Complex from A . vinelandii
So,5 values of pyruvate over the whole pH range studied. In line with tne data already presented, the effectors, apart from producing the shift in alkaline direction of pKF, shift in addition the pKF and pK; values. It is of interest to mention that AMP and sulphate when tested in the ferricyanide and OJinked reactions enhance the activity at about pH 8.5 but not at pH 7.5. Fig.5 gives the dependence of h on the pH. It is clear that h is determined by subunit ionization within the ES complex (cf. log V versus pH). Most of the coefficients are above 2, suggesting the involvement of interactions between more than two subunits, either identical or not. These results rule out the multi-site hypothesis as proposed by Atkinson et al. [23]. According to Dixon and Webb [22] the pHdependence of the rate equation is:
\ LV-
-
+ -
6
7 PH
8
+
9
Fig. 5. Dependence of ( A ) log V, ( B ) P S ~ and . ~ ( C ) h .for pyruvate on the p H . Each point was derived from plots as shown in Fig.2 under the conditions described. Measurements were performed in 50mM Tris-acetate. Details are given in the text. (M No ) additions; ( x ~ - -x-) in the presence of 0.1 mM sulphate; (A---A) in the presence of 0.1 mM AMP
tion, the pH of each cuvette was measured. Corrections were made for irreversible destruction of the enzyme by a change of pH. This was tested by exposing the enzyme to the range of pH values and measuring the activity after readjustment of the pH to a standard value (pH 7.4). At pH values more extreme than used here the enzyme nearly completely denatures. In Fig. 5 the relation between log V or pSo,, with the pH with or without effectors are given. V was extrapolated from reciprocal plots and So,5 values were derived from Hill plots. According to Dixon and Webb [22] the two breaks, at pH 6.7 and pH 8.0 in the curve log V versus pH represent pK-values of the 'enzyme-substrate complex', KFs, at the acid side, at the alkaline side. Below pKfS, and above pKY, log V decreases (slopes 0.8 and 1 respectively). Moreover AMP and sulphate shift pKY towards a higher value, thus preventing deprotonation. Upon increasing the AMP concentration to 2 mM (not shown), K F even seems to disappear. Assuming that pK, is identical with pSo,, the analysis of the pS,,, versus pH graphs, shows in addition to the p p s values, two pK values at pH 6.2 (pKF) and at pH 7.2 (pK:) evidently due to ionizations in the enzyme. The slopes of the straight-line sections are approximately integrals (0 and - 1). AMP and sulphate cause a decline of the
[sl s0.5
For an enzyme with h interacting sites (assuming that the concentration of ES complexes that contain less than h molecules of substrate is negligibly small) Eqn (1) must be modified (cJ[23]) to:
For the Hill plot :
es, showing that
/I
is independent of the pH in the multi-
site hypothesis. Control of Activity by the Acetyl-CoAICoA and NADHINADt Ratio It was reported by Bresters et al. [24,25] that allosteric control of the reaction can be obtained by the strong inhibitory action of acetyl-CoA (Ki = 8 pM). Allosteric inhibition is only observed in the presence of phosphate for two reasons: (a) like AMP, phosphate, as an acetyl-CoA antagonist, has an activating
35 1
T. W. Breaters. A . dc Kok. and C. V e e p
effect ; (b) the presence of phosphotransacetylase, converting acetyl-CoA into acetylphosphate, relievcs part of the inhibition. In the absence of phosphate or in preparations free of phosphotransacetylase only normal inhibitory patterns by acetyl-CoA are observed [24]. In addition it was shown [24,25] that the presence of a CoA-regenerating system relieved acetyl-CoA inhibitions as a cause for S-shaped activity-CoA curves. In fact it is interesting to mention that in the absence of a CoA-regenerating system hyperbolic activity-CoA curves can also be found in the cases where the activity is measured on the Aminco-Chance dual-wavelength spectrophotometer or fluorimetrically. This is due to the fact that with these more sensitive techniques, initial velocities can be measured more reliably at low CoA concentrations, whereas with the normal type of spectrophotometer a relatively large proportion of the CoA is converted during the time a small increase in NADH concentration is measured. Nevertheless the fact remains that apart this instrumental artefact, the pyruvate-NAD+ activity is controlled by the acetyl-CoA/CoA ratio. , By variation of the concentration of NAD', attention is focussed on the third enzyme of the complex, lipoamide dehydrogenase. Studies have been performed to elucidate the reaction mechanism of this enzyme [26,27]. Fig.6 illustrates the need to use a CoA-regenerating system for experiments performed on a Zeiss PMQ I1 spectrophotometer to ensure that real initial velocities are measured. The stimulation of the activity, compared with the measurements in Tris-HC1, suggests that the activity observed in the presence of phosphate and in the absence of a CoA-regenerating system is due to diminished inhibition by acetyl-CoA produced. .The fact that phosphate is inhibitory with respect to the reaction in Tris-HC1, in the presence of a CoAregenerating system could at first glance be due to a lowering of the M 8 + concentration to phosphate, which in terms lowers the Mg2+ TPP concentration from about 0.4 mM inTris to about 0.1 mM in 5 mM phosphate. On the other hand phosphate has an activating effect like AMP and under the conditions of this experiment the K,,, for M$+ .TPP is approximately 15 pM. Thus the inhibition must be due to a different effect. One of the possibilities is the highaffinity metal-ion site (one site per flavin) as found by Mn2+-bindingstudies [21]. The K , for NAD' remains unchanged under all conditions employed and was found to be 0.1 mM. It is about the same as that (0.18 mM) for pure lipoamide dehydrogenase from the same organism [26] and decreases at higher pH values. The inhibition by NADH is competitive with respect to NAD and depends somewhat on the buffer used. Ki = 40 pM in the absence of phosphate and +
c ._
E
0
10 l / ( N A D * ] (rnM-')
Fig.6. Dependence of the rate of NADH production on dfferenl conditions. Lineweaver-Burk plots of the rate measured in the presence of 4 m M pyruvate, 2 mM M$+, 1.5 mM dithiothreitol, 0.1 mM CoA, 0.5 rnM TPP and buffer pH 7.5. The complex was c o n t a y n a t e d with phosphotransacetylase activity. No regenerating system present and either 50 rnM Tris-HCI (00) or 50 mM potassium phosphate buffer (A-A); in the presence of a CoAregenerating system (30 m M oxaloacetate plus 2.5 units dialyzed 0 ) or 50 mM citrate synthase) and either 50 mM Tris-HC1 (6 Temperature: 25 'C potassium phosphate buffer (A-A).
20 pM in this presence; the latter value is slightly higher than previously reported [24]. In addition, NADPH is a weak competitive inhibitor with respect to NAD' (Ki= 450 pM in the presence of phosphate). Increased affinity for NADH in the presence of phosphate might be expected in view of competitive action of the phosphate ion on the pig heart enzyme [28]. Control of the Activity b y the Energy Charge
It was shown that AMP and ADP stimulate the activity of the complex at low pyruvate concentrations, but not specifically. ATP is a weak competitive inhibitor with respect to NAD' (Ki= 1.4 mM). No evidence was obtained that the activity is regulated by phosphorylation and dephosphorylation [29,30]. The activity, like the E. coli complex [8], is sensitive towards the energy charge [13]. At low pyruvate concentration the activity reaches an optimum before decreasing (Fig. 7). However, there is only slight inhibition at maximum charge compared with the activity without added nucleotides. In experiments where the M$+ concentration is decreased from 10 to 1 mM, the decrease of the curve at high energy charge is even sharper, probably due to chelation by ATP of the metal necessary for the reaction. Lowering of the concentration of nucleotides does not affect the results significantly. Also seen in Fig. 7 is the influence of the negative effector acetyl-CoA. Since these experiments can, at low pyruvate concentrations, be simply explained by the change
Regulation of Pyruvate-Dehydrogenase Complex from A . vinelandii
352
Energy charge
Fig. 7. Effect of' energy charge on the pj'ruvutr c/rhj~diogcnasecornplex f r o m A. vinelandii. The experiments were carried out in the presence of 10 mM M g z + , 1.5 mM dithiothreitol, 0.1 mM CoA, 0.5 m M TPP, 50 mM Tris-HC1 (pH 7.5). 1 m M NAD' and pyruvate as indicated. Desired values of the energy chargc were obtained with varying amounts of ATP, ADP and A M P (total concentration 5 mM) and 3.5 units dialyzed myokinase ( p H 7.5). Temperature: 2 5 - C . The activity of the complcx without added nucleotides is given at the ordinate. The values are expressed a s a percentage of the highest activity obtained in these experiments (7 limo1 x ) 0.5 mM pyruvate; NADH produced min-' mg-'). ( x (M) 0.5 mM pyruvate plus 0.1 mM acetyl-CoA; (A-A) 5 mM pyruvate ~
in affinity of the complex for pyruvate induccd by AMP, experiments were also performed at higher pyruvate concentration. The curve shows that increase in activity at low energy charge and a marked inhibition at high energy charge is observed as compared with the activity without added nucleotides. A similar curve was obtained by Veeger et al. [25] by measuring the energy charge at low pyruvate concentration and in the presence of 1 mM sulphate, which condition was introduced by using undialyzed myokinase. The results with the pyruvate dehydrogenase complex of A . vinelundii show that the totally different way of regulation of the E.coli complex [8,31] with respect to complexes from other sources (mammalian, potato and Neurospova crassa) [16,18,32-341, is not unique. The bacterial complexes show a S-shaped activity- [pyruvate] relation, stimulation by AMP and sulphate (which has been observed only with the . A . vinelandii complex), competitive inhibition by acetyl-CoA with respect to pyruvate, regulation by energy charge. The other complexes show a hyperbolic activity- [pyruvate] relation : no stimulation by AMP, inhibition by acetyl-CoA by competition with CoA and regulation by phosphorylation - dephosphorylation [5 - 7,29,30,35 - 371. In view of these facts it seems that large differences exist between the type of regulation of the eukaryotic
and prokaryotic systems. Another interesting difference between the systems exists. In the mammalian complex the two pyruvate dehydrogenase chains do not seem to be completely identical, since only one chain can be phosphorylated; furthermore the two flavins of lipoamide dehydrogenase are in different environments [38], also within the complex [39]. In purified lipoamide dehydrogenase from A . vinelandii the two flavins seem to have identical micro-environments as judged from fluorescence lifetime studies ; within the complex however small differences are visible [39]. Two main differences exist between the A . vinelandii and E. coli complexes : (a) the effect of effectors on the Hill coefficient of the activity- [pyruvate] relation; no effect is induced in the A . vinelundii cohplex by either AMP or acetyl-CoA, in contrast to the results found with the E. coli complex [31]; (b) the absence of any effect of phosphornolpyruvate on the inhibition by acetyl-CoA of the A . vinelandii complex in contrast to the relief of inhibition of the E. coli complex [4,8]. It can be concluded from the pH-activity relation that an unknown group with pK = 8.0 is involved in catalysis and cooperativity. When this group is in the protonated form the complex is fully active and maximum cooperativity is obtained. Positive eflectors shift the pK to a higher value, lower pS,,, of pyruvate and enhance equally the maximum activity of the ferricyanide and oxygen-linked activities of thc first enzyme. It is therefore likely that this group (probably an -SH group) is located in the first cnzyme and involved in the transfer of the hydroxyethyl group from TPP to protein-bound lipoate. Bisswanger and Henning [31] proposed that cooperativity of pyruvate binding leads to increased interactions between the subunits of the E. coli complex. Their experiments are based on measurements of the activity. Our data, especially the pH-dependence of the overall activity, combined with the absence of cooperative kinetics in the oxidase rcaction ( c ; t [ll]) and the constant Hill coefficient in the presence of positive and negative effectors, show that at least for the A . vinelandii enzyme a different explanation is valid. The pH studies show that the cooperative kinetics are connected with the pyruvate-containing (ES) complex and not with pyruvate binding by the free complex. The observations exclude the allosteric models of Monod [40] and Koshland [20] as models for the cooperative phenomena, as well as the multisite hypothesis of Atkinson [13]. The value of h shows the same pH-dependence as log V . The regulatory properties are thus mediated through the second (transacetylase) and/or the third (lipoamide dehydrogenase) enzyme, an idea confirmed by labelling experiments [21]. Furthermore, the state of reduction of the flavoprotein might contribute to the cooperativity. The flavoprotein has a high catalytic ccntre activity. Con-
353
T. W . Hrestcrs. A. dc Kok. and C. Veeger
sequently at low pyruvate concentration and in contrast to the situation at high pyruvate concentration, the flavoprotein will be in steady-state catalysis mainly in its oxidized form. Since reduction leads to changes in conformational interaction [21] it seems reasonable to relate this with the cooperativity. Although definite proof is still lacking about the possible association of phosphotransacetylase with the complex it is clear that the presence ofthe latter enzyme adds a powerful regulatory tool to the cell system. It is of interest in this connection that E.coli also contains phosphotransacetylase. We wish to thank M r B. J. Sachteleben for drawing the figures. The present investigation was supported by the Netherlands Foundation for Chemical Research (S.O.N.)and with financial aid from the Netherlands Organization for the Advancement of Pure Rcscarch (Z.W.O.).
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18. Harding, R . W., Carline. D. F. & Wagncr. K. P. (1970) Arch. Biochem. Biophgs. 138,653- 661. 19. Poulsen. L. L. & Wedding, K. T. (1970) J. B i d . Chem. 245, 5709- 5711. 20. Koshland, D. E., Nemethy, G. & Filmer, D. (1966) Biodiemisrrv, 5, 365 - 385. 21. Grande, H. J., Bresters, T. W., De Abreu, R . A., dc Kok. A. & Veeger. C. (1975) Eur. J. Biochem. 59, 355-363. 22. Dixon, M. & Webb, E. C. (1964) in The Enzymes, Longmans, London. 23. Atkinson. D. E., Hathaway, J . A. & Smith, E. C. (1965) J. Biol. Chtm. 240, 2682 2690. 24. Brestcrs, T. W., Krul. J., Scheepens, P. & Veeger. C . (1972) FEBS Lett. 22, 305- 309. 25. Veeger, C., Krul, J., Bresters, T. W., Haaker, H., Wassink J. H., Santema, J. S . & de Kok, A. (1971) in .Sti.uc./ure und Funcrion of Enrgmcs (J. Drenth, R. A. Oosterbaan & C. Veeger, eds) vol. 29, pp. 217- 234. North-Holland, American Elsevier, Amsterdam. 26. Van den Broek. H. W. J. (1971)Thesis, Agricultural University. Wageningen, Mededelingen Landbouwhogeschool, Wageningcn. pp. 71 - 78. 27. Van Muiswinkel-Voetberg, 11. ( 1 972) Thesis, Agricultural University, Wagcningen, Mededelingen Landbouwhogcschool, Wageningen, pp. 72 - 74. 28. Veeger. C. & Massey. V. (1962) Biochim. Biophys. Acra, 64. 83-100. Hartman, U. & Siess. E. (1972) FEBS Lett. 27, 29. Wieland, 0.. 240 - 244. 30. Wicland, O., Patzelt, C . & Liimer, G. (1972) Eur. J. Biochrm. 26,426 - 433. 31. Bisswanger, H. & Henning, U. (1971) Eur. J . Biochem. 24, 376- 384. 32. Bremer, J. (1969) Eur. J . Biochem. 8, 535-540. 33. Hucho. F., Randall, D. D., Roche. T. E., Burgett, M. W., PelIcy, J. W. & Reed, L. J. (1972) Arch. Biochmi. Biophys. 151, 328 - 340. 34. Crompton, M. & Laties, G. G. (1971) Arch. Biochrm. Biophys. 143, 143 - 150. 35. Barrera. C. R., Namihira, G., Hamilton, L., Munk. P., Eley, M. H., Linn, T. C. & Reed, L. J . (1972) .Arch. Biochem. Bioph1.S. 148, 343- 358. 36. Wieland, O., von Jagow-Westcrmann, B. & Stuckowski, B. (1 969) Hoppe-Sevler’s Z. Physiol. Chem. 350, 329. 37 Tsai, C. S.,Burgett, M. W. & Reed, L. J. (1973) J . Biol. Chem. 248,8348-8352. 38 Wahl, P.. Auchet, J. C., Visser. A. J. W. G. &Veeger, C. (1975) Lur. .I. BiocAem. 50. 413-418. 39 Veeger, C., Visser. A. J. W. G., Krul, J.. Grande. H. J.. De Abreu, R. A. & de Kok, A. in Proc. 5 / h Sytnpo.sium o t i Flusins and Flusoproreins (T. P. Singer, ed.) Elsevier. Amsterdam, in press. 40 Monod, J . , Wyman, J. & Changeux, J. P. (1965) J . Mol. Biol. 12. 88-118. -
T. W. Bresters. A. de Kok, and C. Vecgcr, Laboratorium voor Biochemie der Landbouwhogeschool. De Dreijen 11, Wageningen, The Netherlands
The Pyruvate-Dehydrogenase Complex from Azotobacter vinelandii 2. Regulation of the Activity Tjarda W. BRESTERS, Arie DE KOK, and Cees VEEGER Department of Biochemistry, Agricultural University, Wageningen (Received June 25/August 27, 1975)
The presence of activators (AMP and sulphate) or inhibitors (acetyl-CoA) has no influence on the Hill coefficient of the S-shaped [pyruvate] - velocity curve of either the pyruvate-NAD' overall reaction ( h = 2.5) or that of the pyruvate-K,Fe(CN), activity of the first enzyme (11 = 1.3). pH studies indicated that the Hill coefficient is dependent on subunit ionization within the pyruvate-containing complex and not on those in the free complex. It is concluded that pyruvate conversion rather than pyruvate binding is responsible for the allosteric pattern. The activity is, due to absence of a protein kinase, mainly regulated at the acetyl-CoA/ CoA, and NADH/NAD+ levels and by the value of the energy charge.
Differences have been observed between the mechanisms of regulation of the activity of the pyruvate dehydrogenase complexes from mammals and bacteria. The activity is inhibited in both cases by the products of pyruvate oxidation : acetyl-CoA and NADH [l - 41. The inhibition by acetyl-CoA is reversed by CoA in the mammalian system and by pyruvate in the Eschericltia coli system. NADH inhibition is exerted through action of lipoamide dehydrogenase and is competitive with respect to NAD'. The relative importance of product inhibition for the mammalian system remains questionable, since Linn and co-workers [5,6] and Wieland and von Jagow-Westermann [7] described the regulation by a phosphorylation and dephosphorylation process. Phosphorylation and concomitant inactivation is catalyzed by an ATP-specific kinase. The activity is restored by the action of a phosphatase. Kinase and phosphatase are both M&+-dependent. By combined action the activity of the complex can be regulated by the ATP/ADP ratio. Abbreviation. TPP, thiamine pyrophosphate. Enzymes. Pyruvate dehydrogenase complex is pyruvate-NAD'
reductase, decarboxylating, CoA acetylating (EC 1.2.4.1); pyruvate dehydrogenase is pyruvate-K,Fe(CN), reductase, decarboxylating (EC 1.2.2.2); transacetylase or lipoate acetyltransferase (EC 2.3.1.12); lipoamide dehydrogenase (EC 1.6.4.3): phosphotransacetylase or phosphate acetyltransferase (EC 2.3.1.8); citrate synthase (EC 4.1.3.7); carnitine acetyltransferase (EC 2.3.1.7); myokinase or adenylate kinase (EC 2.7.4.3).
Such a type of regulation does not exist in E. coli [8]. Shen et ul. [9,10] proposed that the complex of E.coli is subject to regulation by the energy charge and also to the concentration of negative feedback effectors. The enzyme pyruvate dehydrogenase which catalyzes the first step of the overall process seems to be the main site for the regulation by energy charge. In this paper the regulation of the Azotobucter vinelandii complex is described and compared with the properties of complexes from other sources.
MATERIALS AND METHODS Materials and the methods for the determination of the activity of the partial reactions of the enzymes of the complex as well as for the overall reaction are given in the preceding paper [ll]. The pyruvate dehydrogenase complex used in these studies was purified up to step4 and had a specific activity of 7 units . mg protein-'. It contained some phosphotransacetylase. The CoA concentration was calculated from - SH group measurements according to Ellman [12] and the acetyl-CoA concentration with a system containing oxaloacetate, citrate synthase and 5,5'-dithiobis(2nitrobenzoic acid) ( E = 13600 M-' cm-l at 412 nm, pH 8.0). The CoA-regenerating systems used consisted either of 0.2 mM oxaloacetate plus 3.5 enzyme units
348
Regulation of Pyruvate-Dehydrogenase Complex from A . vinelundii
of citrate synthase or 20 mM carnitine plus 2 enzyme units of carnitine acetyltransferase. Desired values of the energy charge of the adenylate pool were obtained with the aid of myokinase after mixing the proper concentrations of AMP, ADP and ATP [13]. An equilibrium constant of 2.26 (10 mM MgCl,, pH 7.4, 25°C) was used for the reaction 2 ADP eATP AMP according to Bergmeyer [ 141.
/
+
RESULTS AND DISCUSSION Efiect of M g 2 + . T P P on the Activity
The activity of purified preparations from different sources are partially dependent on M$+ or Ca2+ as cofactor [15- 171, whereas these metals are not required for the activity of the complex from Neurospora c r a m [ 181. Purified pyruvate dehydrogenase complex from A . vinelandii contains small amounts of Mg2+ as judged from the low residual activity, when no metal was added to the reaction mixture; EDTA inhibits this activity. Frequently MnZ+ can be used in M$+dependent reactions. From the influence of the metal concentration on the overall activity, measured in 0.05 M Tris-HC1 (pH 7.6) under standard conditions, the K , values for MgZf and Mn2+ were found to be 100 pM and 10 pM respectively (Fig. 1). CaZ+acts as a competitive inhibitor with respect to Mg2+ ( K i = lOpM) and does not induce activity in the absence of M$+. A K, = 25 pM was obtained for the Mgzf .TPP complex varying the concentration of TPP in the presence of 10 mM MgZ (cJ [9]). At higher pyruvate concentrations (10 mM), the K,,, for M$+ . TPP decreases to 10 pM. Thus it is clear that the K, for Mgz+ as determined from Fig. 1 does not reflect the affinity of the enzyme for Mg". Under the conditions of this experiment the K , for M$+ .TPP is 50 pM, in good agreement with the values found at high Mg" concentrations. F i g 1 also shows that the dependence of thc activity on the Mg2+-TPP concentration is greatly influenced by the presence of AMP and the reaction product acetyl-CoA. Sulphate, and to a less extent phosphate, enhance like AMP the affinity of the enzyme for M$+ . TPP. Acetyl-CoA decreases the affinity and behaves as a competitive inhibitor (Ki= 10 pM) with respect to pyruvate (c$ [ll]).
50 1/ [ Mg2'
0
100
. TPP] (rnM-')
Fig. 1. Effect of M$ and Mg2 . T P P on the overall octivity of' the pyruvate dehydrogenase complex of' A. vinelandii in the absence or presence of' ucetyl-CoA and A M P respectively. Reaction mixture as described [ l l ] in the presence of 5 mM pyruvate, 0.5 mM TPP and 0.05 M Tris-HCI. The final pH was 7.6. The M$+ concentrations arc indicated in the figure. The concentrations Mg2+. TPP are calculated using a Kd of 0.41 mM for the M$+ . TPP complex [19].Tempcrature:25"C.(x-x)Noeffectorpresenl;(AA) in the presence of 0.1 mM AMP; (-0) in the presence of 1 niM in thc presence of 10 pM acetyl-CoA AMP; (-0) +
+
+
Dependence of the Activity on the Pyruvate Concentration
Fig.2 shows the rate of the overall reaction at different pyruvate concentrations. Pyruvate exerts a positive homotropic effect. AMP and sulphate stimulate the activity at lower pyruvate concentration. Acetyl-CoA inhibits competitively; this inhibition can
0
0
1
2
3
4
5
6
7
8
9
10
co
[Pyruvate] (mM)
Fig.2. Depeiidence of the rate of thr ovc+-all reac,iiorr on the pyruvate concentrution in the presence or abstwcr ~flecrors. Reaction mixture as described [ l l ] in the presence of 5 mM Mg2+. 0.5 mM TPP and 0.05 M Tris-HCI, final pH 7.5, at 27°C. The production of NADH was measured on the Aminco-Chance dualwavelength spectrophotometer (380- 345 nm) using the 5-20 ?,; A ) in the prestransmission scale. (U No) additions: (A in thc presence of 0.1 mM ence of 10 pM acetyl-CoA; (-) AMP; ( x ~x ) in the presence of 0.1 mM sulphate
be reversed by the positive effectors. It is convenicnt in allosteric proteins to use the substrate concentration at half saturation ( S o , s ) ,since K , cannot be calculated from curves which dcviate from MichaelisMenten kinetics [20]. So.5 = 1.9 mM for pyruvate under the conditions used. In Fig. 3 a Hill plot of the same results is presented. Maximum velocities were extrapolated from the
349
T. W. Bresters. A. de K o k , and C. Veeger
Table 1. Influence ojrhe eflector concentration on the overall reaction of the pyruvate dehydrogenase complex from A. vinelandii Eo,5, the concentration of effector required for half maximal effect, was determined from a curve of reaction rate as a function of the effector concentration at the pyruvate and Mg2+ . TPP concentrations indicated. All curves were hyperbolic Effector
Type of action'
+ +
AMP ADP
+
Acetyl-CoA
-
so:-
Pyruvate
M2'
2 2 0.4 0.16
0.5
60
0.5
160 140
.TPP
0.4 0.4
8
+ is stimulation, - is inhibition.
Fig. 3. Hill plots of the data pwsented in Fig. 2. The same symbols wcre uscd; details, see text
l/velocity V E ~ S U Sl/[pyruvate] curves. The Hill coefficients ( h ) as calculated from the plots are in all cases 2.6-2.7, thus it must be concluded that the cooperativity is not influenced by the presence of the effectors. The shapes of the curves are not dependent on the protein concentration. The effects as observed with AMP and sulphate in stimulating the activity and reversing the acetylCoA inhibition at low pyruvate concentrations are also exerted with decreasing efficiences by ADP, GMP, GDP, phosphate, arsenate, maleate, fumarate and malonate. ATP is slightly inhibitory at 5 mM concentration. The stimulation by AMP or sulphate is not dependent on the presence of sulfhydryl compounds like dithiothreitol in the reaction mixture. In the absence, however, I/ is about 202, lower either with or without the effector (cJ[lo]). In Table 1 the influence of some of the strongest effectors is summarized. In using the alternative electron acceptor ferricyanide to locate the influence of the effectors on the first enzyme of the complex, the effects of AMP and acetyl-CoA are still present and compete with each other or with pyruvate. Flow of electrons to ferricyanide via the lipoyl moieties of the second enzyme will not occur as can be concluded from experiments in which the lipoyl moieties are blocked with N-ethylmaleimide [21]. Fig. 4 shows that the Hill coefficients are 1.3(k0.2). At least part of the characteristics seem to be present
-1.6
-0.8
0
0.8
log [Sl (mM)
Fig. 4. Hill plots of the ferric.).anide-lirihe(~pyruvate deliydrogmuse uctiviry. The activity was measured in Tris-HCI as described [I 11 with the whole complex. The pyruvate concentrations were as indicated. 1' was obtained from Lincwcavcr-Burk plots. ( A ----A) No effector present; ( x -x ) in the presence of 10 pM acetylCoA; (M) in the presence of 20 pM acetyl-CoA; (M) in the presence of 1 mM AMP
in the ferricyanide-linked pyruvate dehydrogenase (partial) reaction. On the other hand one could argue whether, although consistent, such a deviation from h = 1.O is significant. Especially in view of the observation that in the pyruvate oxidase reaction. on which AMP and acetyl-CoA also exert their typical effects, no cooperative effects by pyruvate are observed
(4[111). The Influence of the p H the Rate of the Overall Reaction
OII
The influence of the pH on the velocity was studied with 1 mM NAD' and varying pyruvate concentrations at several pH values. Before and after the reac-
350
Regulation of Pyruvate-Dehydrogenase Complex from A . vinelandii
So,5 values of pyruvate over the whole pH range studied. In line with tne data already presented, the effectors, apart from producing the shift in alkaline direction of pKF, shift in addition the pKF and pK; values. It is of interest to mention that AMP and sulphate when tested in the ferricyanide and OJinked reactions enhance the activity at about pH 8.5 but not at pH 7.5. Fig.5 gives the dependence of h on the pH. It is clear that h is determined by subunit ionization within the ES complex (cf. log V versus pH). Most of the coefficients are above 2, suggesting the involvement of interactions between more than two subunits, either identical or not. These results rule out the multi-site hypothesis as proposed by Atkinson et al. [23]. According to Dixon and Webb [22] the pHdependence of the rate equation is:
\ LV-
-
+ -
6
7 PH
8
+
9
Fig. 5. Dependence of ( A ) log V, ( B ) P S ~ and . ~ ( C ) h .for pyruvate on the p H . Each point was derived from plots as shown in Fig.2 under the conditions described. Measurements were performed in 50mM Tris-acetate. Details are given in the text. (M No ) additions; ( x ~ - -x-) in the presence of 0.1 mM sulphate; (A---A) in the presence of 0.1 mM AMP
tion, the pH of each cuvette was measured. Corrections were made for irreversible destruction of the enzyme by a change of pH. This was tested by exposing the enzyme to the range of pH values and measuring the activity after readjustment of the pH to a standard value (pH 7.4). At pH values more extreme than used here the enzyme nearly completely denatures. In Fig. 5 the relation between log V or pSo,, with the pH with or without effectors are given. V was extrapolated from reciprocal plots and So,5 values were derived from Hill plots. According to Dixon and Webb [22] the two breaks, at pH 6.7 and pH 8.0 in the curve log V versus pH represent pK-values of the 'enzyme-substrate complex', KFs, at the acid side, at the alkaline side. Below pKfS, and above pKY, log V decreases (slopes 0.8 and 1 respectively). Moreover AMP and sulphate shift pKY towards a higher value, thus preventing deprotonation. Upon increasing the AMP concentration to 2 mM (not shown), K F even seems to disappear. Assuming that pK, is identical with pSo,, the analysis of the pS,,, versus pH graphs, shows in addition to the p p s values, two pK values at pH 6.2 (pKF) and at pH 7.2 (pK:) evidently due to ionizations in the enzyme. The slopes of the straight-line sections are approximately integrals (0 and - 1). AMP and sulphate cause a decline of the
[sl s0.5
For an enzyme with h interacting sites (assuming that the concentration of ES complexes that contain less than h molecules of substrate is negligibly small) Eqn (1) must be modified (cJ[23]) to:
For the Hill plot :
es, showing that
/I
is independent of the pH in the multi-
site hypothesis. Control of Activity by the Acetyl-CoAICoA and NADHINADt Ratio It was reported by Bresters et al. [24,25] that allosteric control of the reaction can be obtained by the strong inhibitory action of acetyl-CoA (Ki = 8 pM). Allosteric inhibition is only observed in the presence of phosphate for two reasons: (a) like AMP, phosphate, as an acetyl-CoA antagonist, has an activating
35 1
T. W. Breaters. A . dc Kok. and C. V e e p
effect ; (b) the presence of phosphotransacetylase, converting acetyl-CoA into acetylphosphate, relievcs part of the inhibition. In the absence of phosphate or in preparations free of phosphotransacetylase only normal inhibitory patterns by acetyl-CoA are observed [24]. In addition it was shown [24,25] that the presence of a CoA-regenerating system relieved acetyl-CoA inhibitions as a cause for S-shaped activity-CoA curves. In fact it is interesting to mention that in the absence of a CoA-regenerating system hyperbolic activity-CoA curves can also be found in the cases where the activity is measured on the Aminco-Chance dual-wavelength spectrophotometer or fluorimetrically. This is due to the fact that with these more sensitive techniques, initial velocities can be measured more reliably at low CoA concentrations, whereas with the normal type of spectrophotometer a relatively large proportion of the CoA is converted during the time a small increase in NADH concentration is measured. Nevertheless the fact remains that apart this instrumental artefact, the pyruvate-NAD+ activity is controlled by the acetyl-CoA/CoA ratio. , By variation of the concentration of NAD', attention is focussed on the third enzyme of the complex, lipoamide dehydrogenase. Studies have been performed to elucidate the reaction mechanism of this enzyme [26,27]. Fig.6 illustrates the need to use a CoA-regenerating system for experiments performed on a Zeiss PMQ I1 spectrophotometer to ensure that real initial velocities are measured. The stimulation of the activity, compared with the measurements in Tris-HC1, suggests that the activity observed in the presence of phosphate and in the absence of a CoA-regenerating system is due to diminished inhibition by acetyl-CoA produced. .The fact that phosphate is inhibitory with respect to the reaction in Tris-HC1, in the presence of a CoAregenerating system could at first glance be due to a lowering of the M 8 + concentration to phosphate, which in terms lowers the Mg2+ TPP concentration from about 0.4 mM inTris to about 0.1 mM in 5 mM phosphate. On the other hand phosphate has an activating effect like AMP and under the conditions of this experiment the K,,, for M$+ .TPP is approximately 15 pM. Thus the inhibition must be due to a different effect. One of the possibilities is the highaffinity metal-ion site (one site per flavin) as found by Mn2+-bindingstudies [21]. The K , for NAD' remains unchanged under all conditions employed and was found to be 0.1 mM. It is about the same as that (0.18 mM) for pure lipoamide dehydrogenase from the same organism [26] and decreases at higher pH values. The inhibition by NADH is competitive with respect to NAD and depends somewhat on the buffer used. Ki = 40 pM in the absence of phosphate and +
c ._
E
0
10 l / ( N A D * ] (rnM-')
Fig.6. Dependence of the rate of NADH production on dfferenl conditions. Lineweaver-Burk plots of the rate measured in the presence of 4 m M pyruvate, 2 mM M$+, 1.5 mM dithiothreitol, 0.1 mM CoA, 0.5 rnM TPP and buffer pH 7.5. The complex was c o n t a y n a t e d with phosphotransacetylase activity. No regenerating system present and either 50 rnM Tris-HCI (00) or 50 mM potassium phosphate buffer (A-A); in the presence of a CoAregenerating system (30 m M oxaloacetate plus 2.5 units dialyzed 0 ) or 50 mM citrate synthase) and either 50 mM Tris-HC1 (6 Temperature: 25 'C potassium phosphate buffer (A-A).
20 pM in this presence; the latter value is slightly higher than previously reported [24]. In addition, NADPH is a weak competitive inhibitor with respect to NAD' (Ki= 450 pM in the presence of phosphate). Increased affinity for NADH in the presence of phosphate might be expected in view of competitive action of the phosphate ion on the pig heart enzyme [28]. Control of the Activity b y the Energy Charge
It was shown that AMP and ADP stimulate the activity of the complex at low pyruvate concentrations, but not specifically. ATP is a weak competitive inhibitor with respect to NAD' (Ki= 1.4 mM). No evidence was obtained that the activity is regulated by phosphorylation and dephosphorylation [29,30]. The activity, like the E. coli complex [8], is sensitive towards the energy charge [13]. At low pyruvate concentration the activity reaches an optimum before decreasing (Fig. 7). However, there is only slight inhibition at maximum charge compared with the activity without added nucleotides. In experiments where the M$+ concentration is decreased from 10 to 1 mM, the decrease of the curve at high energy charge is even sharper, probably due to chelation by ATP of the metal necessary for the reaction. Lowering of the concentration of nucleotides does not affect the results significantly. Also seen in Fig. 7 is the influence of the negative effector acetyl-CoA. Since these experiments can, at low pyruvate concentrations, be simply explained by the change
Regulation of Pyruvate-Dehydrogenase Complex from A . vinelandii
352
Energy charge
Fig. 7. Effect of' energy charge on the pj'ruvutr c/rhj~diogcnasecornplex f r o m A. vinelandii. The experiments were carried out in the presence of 10 mM M g z + , 1.5 mM dithiothreitol, 0.1 mM CoA, 0.5 m M TPP, 50 mM Tris-HC1 (pH 7.5). 1 m M NAD' and pyruvate as indicated. Desired values of the energy chargc were obtained with varying amounts of ATP, ADP and A M P (total concentration 5 mM) and 3.5 units dialyzed myokinase ( p H 7.5). Temperature: 2 5 - C . The activity of the complcx without added nucleotides is given at the ordinate. The values are expressed a s a percentage of the highest activity obtained in these experiments (7 limo1 x ) 0.5 mM pyruvate; NADH produced min-' mg-'). ( x (M) 0.5 mM pyruvate plus 0.1 mM acetyl-CoA; (A-A) 5 mM pyruvate ~
in affinity of the complex for pyruvate induccd by AMP, experiments were also performed at higher pyruvate concentration. The curve shows that increase in activity at low energy charge and a marked inhibition at high energy charge is observed as compared with the activity without added nucleotides. A similar curve was obtained by Veeger et al. [25] by measuring the energy charge at low pyruvate concentration and in the presence of 1 mM sulphate, which condition was introduced by using undialyzed myokinase. The results with the pyruvate dehydrogenase complex of A . vinelundii show that the totally different way of regulation of the E.coli complex [8,31] with respect to complexes from other sources (mammalian, potato and Neurospova crassa) [16,18,32-341, is not unique. The bacterial complexes show a S-shaped activity- [pyruvate] relation, stimulation by AMP and sulphate (which has been observed only with the . A . vinelandii complex), competitive inhibition by acetyl-CoA with respect to pyruvate, regulation by energy charge. The other complexes show a hyperbolic activity- [pyruvate] relation : no stimulation by AMP, inhibition by acetyl-CoA by competition with CoA and regulation by phosphorylation - dephosphorylation [5 - 7,29,30,35 - 371. In view of these facts it seems that large differences exist between the type of regulation of the eukaryotic
and prokaryotic systems. Another interesting difference between the systems exists. In the mammalian complex the two pyruvate dehydrogenase chains do not seem to be completely identical, since only one chain can be phosphorylated; furthermore the two flavins of lipoamide dehydrogenase are in different environments [38], also within the complex [39]. In purified lipoamide dehydrogenase from A . vinelandii the two flavins seem to have identical micro-environments as judged from fluorescence lifetime studies ; within the complex however small differences are visible [39]. Two main differences exist between the A . vinelandii and E. coli complexes : (a) the effect of effectors on the Hill coefficient of the activity- [pyruvate] relation; no effect is induced in the A . vinelundii cohplex by either AMP or acetyl-CoA, in contrast to the results found with the E. coli complex [31]; (b) the absence of any effect of phosphornolpyruvate on the inhibition by acetyl-CoA of the A . vinelandii complex in contrast to the relief of inhibition of the E. coli complex [4,8]. It can be concluded from the pH-activity relation that an unknown group with pK = 8.0 is involved in catalysis and cooperativity. When this group is in the protonated form the complex is fully active and maximum cooperativity is obtained. Positive eflectors shift the pK to a higher value, lower pS,,, of pyruvate and enhance equally the maximum activity of the ferricyanide and oxygen-linked activities of thc first enzyme. It is therefore likely that this group (probably an -SH group) is located in the first cnzyme and involved in the transfer of the hydroxyethyl group from TPP to protein-bound lipoate. Bisswanger and Henning [31] proposed that cooperativity of pyruvate binding leads to increased interactions between the subunits of the E. coli complex. Their experiments are based on measurements of the activity. Our data, especially the pH-dependence of the overall activity, combined with the absence of cooperative kinetics in the oxidase rcaction ( c ; t [ll]) and the constant Hill coefficient in the presence of positive and negative effectors, show that at least for the A . vinelandii enzyme a different explanation is valid. The pH studies show that the cooperative kinetics are connected with the pyruvate-containing (ES) complex and not with pyruvate binding by the free complex. The observations exclude the allosteric models of Monod [40] and Koshland [20] as models for the cooperative phenomena, as well as the multisite hypothesis of Atkinson [13]. The value of h shows the same pH-dependence as log V . The regulatory properties are thus mediated through the second (transacetylase) and/or the third (lipoamide dehydrogenase) enzyme, an idea confirmed by labelling experiments [21]. Furthermore, the state of reduction of the flavoprotein might contribute to the cooperativity. The flavoprotein has a high catalytic ccntre activity. Con-
353
T. W . Hrestcrs. A. dc Kok. and C. Veeger
sequently at low pyruvate concentration and in contrast to the situation at high pyruvate concentration, the flavoprotein will be in steady-state catalysis mainly in its oxidized form. Since reduction leads to changes in conformational interaction [21] it seems reasonable to relate this with the cooperativity. Although definite proof is still lacking about the possible association of phosphotransacetylase with the complex it is clear that the presence ofthe latter enzyme adds a powerful regulatory tool to the cell system. It is of interest in this connection that E.coli also contains phosphotransacetylase. We wish to thank M r B. J. Sachteleben for drawing the figures. The present investigation was supported by the Netherlands Foundation for Chemical Research (S.O.N.)and with financial aid from the Netherlands Organization for the Advancement of Pure Rcscarch (Z.W.O.).
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T. W. Bresters. A. de Kok, and C. Vecgcr, Laboratorium voor Biochemie der Landbouwhogeschool. De Dreijen 11, Wageningen, The Netherlands
