395

Biochimica et Biophysica Acta, 397 (1975) 395--404 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 67563

HUMAN PLATELET 6-PHOSPHOFRUCTOKINASE R E L A T I O N BETWEEN INHIBITION BY Mg " A T P 2 - AND C O O P E R A T I V I T ¥ TOWARDS F R U C T O S E 6-PHOSPHATE AND INVESTIGATIONS ON THE F O R M A T I O N OF A T E R N A R Y COMPLEX

JAN WILLEM N. AKKERMAN, GERTIE GORTER, JAN OVER, JAN J. SIXMA and GERARD E.J. STAAL

Dept. of Haematology, State University Hospital, Utrecht (The Netherlands) (Received February 21st, 1975)

Summary Human platelet 6-phosphofructokinase (EC 2.7.1.11) shows cooperativity towards Fru-6-P and is allosterically inhibited by high Mg " ATP:- concentrations. No relation could be demonstrated between the cooperativity towards Fru-6-P and the inhibition by Mg " ATP ~-. Increasing the concentrations of Mg " ATP 2- only raised the apparent K m values for Fru-6-P, b u t did not change the Hill constants. A possible formation of a Mg " ATP 2- " enzyme " Fru-6-P complex during catalysis was investigated. Our calculations suggest that such a ternary complex is indeed formed during the reaction.

Introduction

Platelet shape change, aggregation and especially release reaction are energy requiring processes [ 1,2]. Part of this energy is provided by glycolysis which is stimulated b y the mentioned platelet functions [3,4]. Changes in the levels of glycolytic intermediates have indicated that phosphofructokinase plays an important regulatory role in the rat platelet [5,6]. We have recently confirmed this for human platelets (Akkerman, J.W.N., Gorter, G., Staal, G.E.J. and Sixma, J.J., unpublished results). More direct information a b o u t the regulatory role o f this enzyme may be gained from investigations of the purified enzyme. 6-Phosphofructokinase (EC 2.7.1.11) catalyzes the conversion of Fru-6-P to Fru-l,6-P:, coupled with the dephosphorylation of ATP to ADP. This catalysis depends on Mg 2÷. In previous articles we have described a purification procedure and shown that the purified enzyme is activated strongly by sulphate [ 7 8 ] . Rather than ATP alone the Mg " ATP 2- complex is the substrate for the

396 enzyme, but high Mg • ATP 2- concentrations axe inhibitory [9]. Free ATP 4increases the allosteric inhibition by Mg • ATP 2- [9]. When ITP was used as a phosphate donor, free Mg2+ stimulated with an optimal effect at a ratio [Mg 2 + ] / [ M g ' I T P 2-] /> 1.0 [9]. In this paper we report the interdependence between the allosteric inhibition by Mg " ATP 2- and the cooperativity towards Fru-6-P. A direct relationship between these phenomena was observed in phosphofructokinase from pig spleen [10], rat t h y m o c y t e s [11] and rabbit muscle [12]. On the other hand, studies with phosphofructokinase from yeast [13,14], Bacillus licheniforrnis [15] and human erythrocytes [16] showed that the cooperativity towards Fru-6-P was n o t dependent on or parallel with, allosteric inhibition by Mg " ATP 2-. Layzer et al. [ l q ] proposed that the reaction catalyzed by red blood cell phosphofructokinase takes place through a Ping Pong mechanism. In contrast Staal et al. [18] proposed the existence of a ternary complex for the same enzyme. Such a Mg " ATP 2- " enzyme " Fru-6-P complex was also found for the enzyme from rabbit muscle [19]. Using the methods of Slater, Koster, Staal and Veeger [18,20--23] we investigated whether this complex was formed also during catalysis by the human platelet enzyme. Materials and Methods The nucleotide phosphates, added as sodium salts, glycolytic intermediates, cofactors and enzymes used for measurement of phosphofructokinase activity, were obtained from Boehringer-Mannheim. All other chemicals used were of analytical grade. Human platelet phosphofructokinase was partially purified as described previously [7]. The enzyme activity was measured by coupling the formation of Fru-l,6-P2 to the a-glycerophosphate dehydrogenase reaction. The oxidation of NADH was followed at 340 nm in a Perkin-Elmer 124 spectrophotometer at 25°C. The assay mixture contained in a final volume of 3 ml: 0.2 M Tris " HC1 (pH 8.1); 6 mM KC1; 0.05 ml dialyzed auxiliary enzymes (fructose diphosphate aldolase, 10 mg/ml; triosephosphate isomerase, 2 mg/ml; glycerophosphate dehydrogenase, 2 mg/ml); 0.2 mM NADH (disodium salt) and the concentrations of substrates of the reaction catalyzed by phosphofructokinase as indicated in Results. MgC12 was present at concentrations of [ M g t o t a l ] = 3 × [nucleotide phosphateto tal]. The levels of the Mg • ATP 2- complex were calculated with the aid of a stability constant of 20 000 M -1 which has been determined for a medium which resembles the assay mixture used by us [24,25]. The Mg " ITP 2- complex was assumed to have an identical stability constant [26]. Complex formation between Mg2÷ and Fru-6-P and between K ÷ and ATP 4- or IT1~ - was considered negligible because of the low stability constants of these complexes [25,27]. The formation of HATP 3-, Mg" H A T P - a n d similax complexes of ITP 4- was neglected since these complexes represent less than 1% of the total nucleotide content at pH 8.1 [28]. The reaction was started by adding 0.02 ml purified human platelet phosphofructokinase. A unit of enzyme activity is defined as the a m o u n t of enzyme catalyzing the formation of 1 #tool of Fru-l,6-P~ per min at 25°C. The various

397 purified p h o s p h o f r u c t o k i n a s e preparations t ha t were tested (n = 5) had a spec. act. o f a b o u t 7 units/mg protein, as measured at 4 mM Fru-6-P, 0.4 mM ATPtotal and 5 mM MgSO4. The protein c o n t e n t was assayed according to L o w ry et al. [29] using crystalline bovine serum albumin as a standard. The influences of ATP and ITP were n o t disturbed by sodium effects and corrections were n o t necessary.

Results Relation between inhibition by Mg" A T P 2- and cooperativity towards Fru-6-P Phosphofructokinases isolated from different sources are characterized by sigmoidal velocity curves with respect to Fru-6-P and allosteric inhibition at high Mg • ATP 2- concentrations. Both the cooperativity towards Fru-6-P and t h e inhibition by Mg " ATP ~- are most p r o n o u n c e d at neutral pH. Unlike other types o f p h o s p h o f r u c t o k i n a s e the platelet e n z y m e still exhibits these properties to a large e x t e n t when the pH is raised to 8.1 on condition that bivalent anions such as sulphate are absent [ 7 , 8 ] . Fig. 1A illustrates the inhibition by Mg" ATP 2- at pH 8.1. The inhibition is c o u n t e r a c t e d by Fru-6-P. The sigmoidal velocity curves for Fru-6-P (Fig. 1B) illustrate the cooperativity towards this substrate. At rising Mg • ATP 2- concentrations and therefore increasing Mg " ATP 2- inhibition the curves seem to be c om e more sigmoidal. F r o m these data it may be concluded that the inhibition by Mg • ATP 2influences the kinetics towards Fru-6-P and vice versa. We investigated this relationship by comparing the velocity curves for Fru-6-P as measured in the presence and in the absence of inhibition by Mg " ATP 2-. It is generally assumed t hat the inhibition at high Mg " ATP 2- levels involves an inhibitory site which is highly specific for this nucleotide. At pH 8.1 the platelet e n z y m e also shows this specificity [ 7 ] . When Mg • ITP 2- was used as a phosphate d o n o r no inhibition was observed (Fig. 2A). This suggested t h a t Mg " ITP 2- only acted on the catalytic site. Involvement of bot h inhibitor and catalytic site (using ATP) could thus be separated from participation of the catalytic site alone (using ITP). In the absence of inhibition the cooperative interactions with respect to Fru-6-P remained present (Fig. 2B). This was confirmed by calculations of the Hill coefficients: values o f 2.2 were calculated which were i ndependent on the concentrations o f Mg • ITP 2- in the range 0.04--1.98 mM Mg " ITP 2- (Fig. 3). When Mg " ATP 2- was used the Hill values were slightly higher (n = 3.0). Under o u r experimental conditions, again this value was not d e p e n d e n t on the concentrations o f Mg " ATP 2-. Thus, b o t h the absence (Fig. 3B) and the degree (Fig. 3A) o f allosteric inhibition by Mg • ATP 2- had no effect on the cooperativity towards Fru-6-P. In all experiments the n value with respect to Fru-6-P tended to be s o mewh at lower with Mg " ITP 2- than with Mg " ATP 2-. This m ay indicate that binding o f Mg • ITP 2- and of Mg " ATP 2- have a different effect on the cooperativity towards Fru-6-P. The involvement of the inhibitor site for Mg " ATP 2-, however, greatly effected the affinity for Fru-6-P (Table I). Increasing concentrations of the phosphate donors raised the apparent Km values for Fru-6-P. At about 0.5 mM phosphate d o n o r the catalytic site for the nucleotide phosphates appeared t o

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be saturated and n o further e f f e c t o n t h e a f f i n i t y for Fru-6-P was m e a s u r e d w h e n Mg • ITP 2- was used. In contrast, t h e i n v o l v e m e n t o f the i n h i b i t o r y site b e c a m e a p p a r e n t at higher Mg • A T P 2- levels leading t o p r o n o u n c e d i n f l u e n c e s o n a p p a r e n t Km for Fru-6-P values.

A ternary complex in the reaction mechanism of phosphofructokinase? The f o l l o w i n g rate e q u a t i o n for a t w o substrate r e a c t i o n delivers an overall p i c t u r e o f t h e r e a c t i o n c a t a l y z e d b y h u m a n platelet p h o s p h o f r u c t o k i n a s e : Km Fru-6-P

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TABLE

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CONSTANTS

FOR

Fru-6-P WITH AND WITHOUT

INHIBITION

B Y M g - A T P 2-

T h e a p p a r e n t K m v a l u e s f o r F r u - 6 - P i n t h e p r e s e n c e o f M g • A T P 2 - o r M g • I T P 2 - as t h e p h o s p h a t e d o n o r w e r e c a l c u l a t e d f r o m F i g . 3 A a n d F i g . 3 B r e s p e c t i v e l y b y e x t r a p o l a t i o n t o l o g v / ( V ' - v ) = O, w h e r e V ' s t a n d s f o r a p p a r e n t V.

Phosphate (mM)

donor

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K m for Fru-6-P (mM)

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( M g • A T P 2-)

No inhibition

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where E and n stand for phos phof r uct oki nas e and the Hill coefficient, respectively. If the term Km Fru-6-P-E-Mg • ATP 2- is equal to zero, Eqn 1 will represent a Ping Pong mechanism. If this term is unequal to zero a ternary co m p l e x is involved in the reaction [ 1 8 , 2 0 - - 2 3 ] . For calculations of this factor the data f r o m Figs 1 and 2 were replotted in Lineweaver-Burk plots for Fru-6-P (Fig. 4) and Mg " ITP 2- (not shown in a figure). From Fig. 4 apparent Vm ax values at various Mg " ATP 2- (Fig. 4A) and Mg " ITP 2- (Fig. 4B) levels at infinite concentrations of Fru-6-P were derived by extrapolation to [Fru-6-P]-i = 0.

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1 2 3 A p P a r o n t V(zIA rain -1, m1-1 e n z y m e solution) Fig. 5. P l o t s o f a p p a r e n t V v a l u e s ( V a p p . F r u - 6 - P i n E q n 2 ; e x p r e s s e d as ZL4. r a i n -1 • m1-1 e n z y m e s o l u t i o n ) a t c o r r e s p o n d i n g a p p a r e n t K m d a t a f o r F r u - 6 - P ( m M ) ( K m a p p . F r u - 6 - P i n E q n 2 ) u s i n g M g • A T P 2- ( l o w e r l i n e ) a n d M g • I T P 2- ( u p p e r l i n e ) as t h e s e c o n d s u b s t r a t e ( F i g . 5 A ) . S i m i l a r P l o t f o r M g • I T P 2 - u s i n g F r u - 6 - P as t h e s e c o n d s u b s t r a t e s h o w n i n F i g . 5B. V a l u e s c a l c u l a t e d f r o m F i g s 1, 2 a n d 4. P o i n t s c o r r e s p o n d i n g t o i n h i b i t i o n b y M g • A T P 2- w e r e e x c l u d e d f r o m t h e c a l c u l a t i o n s . ( R e g r e s s i o n l i n e s w i t h c o r r e l a t i o n c o e f f i c i e n t (r): F i g . 5 A . y = 0 . 3 2 3 x + 0 . 0 8 6 ; r = 1 . 0 0 f o r M g • A T P 2- a n d y = 0 . 3 7 7 x + 0 . 3 4 3 ; r = 0 . 9 8 f o r M g • I T P 2-. Fig. 5B. y = 0 . 0 7 6 x + 0 . 0 2 9 ; r = 1 . 0 0 ) . S y m b o l s d e f i n e d i n Fig. 1.

T h e s e values were p l o t t e d against t h e respective a p p a r e n t K m values for Fru-6-P (Fig. 5A). T h e lines in Fig. 5A represent t h e graphic n o t a t i o n o f E q n 2, w h i c h can be derived f r o m E q n 1 b y c o n v e r t i n g the latter t o t h e f o r m y = a + b x in w h i c h y = a p p a r e n t Km Fru-6-P and x = a p p a r e n t V at [Fru-6-P] = ~¢ (Vapp Fru-6-P) Km Fru-6-P- E -Mg • A T P 2 Km app Fru-6-P =

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A s s u m i n g that Fru-6-P is t h e first substrate t h e lines o f Fig. 5 A d o n o t pass t h r o u g h t h e origin, w h e t h e r Mg • A T P 2- or Mg " ITP 2- is t h e o t h e r substrate (regression analysis: p < 0.01 using Mg " ITP 2- as p h o s p h a t e d o n o r ) . T h e r e f o r e , t h e t e r m Km Fru-6-P-E-Mg " N T P 2 - / K m Mg • N T P 2- is u n e q u a l t o zero. If Mg • ITP 2- is t h e first substrate Fig. 5B is valid. This line crosses t h e o r d i n a t e at a significant d i s t a n c e f r o m t h e origin {regression analysis p < 0 . 0 1 ) . T h e strong i n h i b i t i o n by Mg ' A T P 2- h a m p e r s similar c a l c u l a t i o n s for this substrate. H o w e v e r , if this i n h i b i t i o n was suppressed b y t h e a d d i t i o n o f sulp h a t e [ 7 ] or c y c l i c 3', 5' AMP [ 3 0 ] results similar t o t h o s e o f Mg " ITP 2- w e r e o b t a i n e d . F u r t h e r m o r e , in the p r e s e n c e o f s u l p h a t e at p H 8.1 t h e platelet e n z y m e s h o w s s t r o n g l y r e d u c e d c o o p e r a t i v i t y t o w a r d s Fru-6-P (n = 1.2) [ 7 ] . U n d e r t h e s e c o n d i t i o n s a ternary c o m p l e x can also be calculated, w h i c h m e a n s

402

that sigmoidal kinetics do n o t disturb these calculations. This is not surprising since in the calculations of the complex the velocity data are extrapolated to infinite Fru-6-P concentration, i.e. to conditions in which n = 1.0. Whatever the first substrate may be these data strongly suggest the involvement of a ternary complex in the reaction mechanism. Discussion Phosphofructokinase isolated from different sources generally shows allosteric inhibition at high Mg " ATP:- levels and cooperativity towards Fru-6-P. The way in which these properties are related, however, is not general. In one group of phosphofructokinases both properties are completely independent and each may be influenced separately by various effectors. Examples of this class are phosphofructokinase from yeast [13,14] Bacillus licheniformis [15] or human erythrocytes [16]. In contrast, the other group of phosphofructokinases shows strong interdependence of these phenomena. The enzyme from pig spleen [10], rat t h y m o c y t e s [11] and rabbit muscle [12] is characterized by a complete loss of cooperativity towards Fru-6-P when Mg " ATP 2- inhibition is absent and furthermore the degree of cooperativity corresponds to the degree of inhibition by Mg " ATP:- (For a recent review see ref. 31). The data presented in this paper show that the human platelet enzyme behaves in the former way i.e. the cooperativity towards Fru-6-P remains present in the absence of Mg • ATP 2- inhibition and this cooperativity is not affected by various degrees of inhibition. Between 0.04 mM and 1.98 mM Mg " ATP 2- the Hill coefficient remained constant (n = 3.0). A similar observation has been made by Kopperschlfiger on yeast phosphofructokinase [32]. Unlike this enzyme which gave Hill values of 1.0 when ITP was present in stead of ATP, the platelet enzyme still exhibits cooperativity towards Fru-6-P when Mg • ITP 2- is the phosphate donor (n = 2.2). Recently, Lee et al. demonstrated that photo-oxidized phosphofructokinase from h u m a n red blood cells looses its cooperativity towards Fru-6-P but still shows the inhibition pattern of ATP [33]. They concluded that cooperativity towards Fru-6-P and inhibition by ATP are independent phenomena and this is well in accordance with the findings presented here for the platelet enzyme• From the data for Mg " ITP 2- a second conclusion may be drawn. In the range of Mg • ITP 2- concentrations used this substrate did not effect the Hill coefficient for Fru-6-P. This indicates that the degree of saturation of the catalytic site for the phosphate donor does not influence the cooperativity towards Fru-6-P. In this respect the platelet enzyme resembles phosphofructokinase from Escherichia coli. Here ATP only functions as a substrate and does n o t inhibit enzyme activity• The various degrees of saturation with ATP had no effect on the cooperativity of this enzyme towards Fru-6-P [34]. Investigations on the reaction mechanism of phosphofructokinase catalysis have been performed using isotope exchange techniques, various substrate analogs [19] or calculations of kinetic data at varying levels of both substrates [18,20--23]. Using the latter m e t h o d the involvement of a ternary complex in the reaction mechanism of the platelet enzyme can be calculated although the

403 reaction mechanism itself remains obscure. In this respect, our findings disagree with those of Layzer et al. [17], who concluded from parallel lines in the reciprocal plots that phosphofructokinase from human red blood cells follows a Ping Pong mechanism. However, the demonstration that the reciprocal plots produce apparent parallel lines is not conclusive evidence for a Ping Pong mechanism [31]. In contrast to Layzer, Staal et al. [18] calculated a ternary complex for the same enzyme using the methods described here for the platelet enzyme. Such a complex was found also in rabbit muscle phosphofructokinase catalysis; in fact the presently available information suggests that a ternary complex is involved in the reaction mechanism of most types of phosphofructokinases [31]. The p o t e n t activities of the free constituents of the Mg • ATP 2- complex already mentioned in the introduction may interfere with formation of the ternary complex thus leading to a more complicated reaction mechanism. The physiological meaning of the results reported here is until now uncertain. The independence of cooperative interactions towards Fru-6-P and allosteric inhibition by Mg " ATP 2- may perhaps provide an extra dimension to regulation phenomena in human platelet glycolysis. But since various ligands similarly effect both properties the netto result might be of relatively minor importance [7,8,30]. Acknowledgements The authors are indebted to Dr E. Borst-Eilers, head of the Blood Transfusion Laboratory, University Hospital Utrecht for supplying platelet suspensions. The statistic evaluation was performed by Dr J.A. Faber, Institute for Mathematical Statistics, University of Utrecht for whose help we are most grateful. The authors also wish to thank Dr J.F. Koster, Department of Biochemistry, Erasmus University, Rotterdam, The Netherlands, for many helpful discussions. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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Human platelet 6-phosphofructokinase. Relation between inhibition by Mg-ATP2-and cooperativity towards fructose 6-phosphate and investigations on the formation of a ternary complex.

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