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Kinetic and physical properties of phosphofructokinase
KINETIC A N D PHYSICAL PROPERTIES OF P H O S P H O F R U C T O K I N A S E TAG E. MANSOUR Department of Pharmacology, Stanford University, School of Medicine, Stanford, California INTRODUCTION STUDIES on different enzymes have established that catalytic activity can be regulated through three main mechanisms. These include: availability of substrate or product to the catalytic site of the enzyme (kinetic control), reversible active-inactive conversion of the enzyme through changes in its molecular structure, and lastly, changes in enzyme activity through conformational alteration induced by a specific ligand on a site other than the catalytic site (allosteric control). It is from this point of view that we have approached our studies on the regulation of phosphofructokinase (ATP:Dfructose-6-P 1-phosphotransferase, EC 2.7.1.11). Early evidence for activation or inhibition of this enzyme was based on changes in the level of the substrate and the products (1-3). A more direct evidence of phosphofructokinase activation was reported during our studies on the regulation of glycolysis in the liver fluke, Fasciola hepatica (4, 5). Incubation of the flukes with serotonin (5-hydroxytryptamine) resulted in a marked increase in phosphofructokinase activity. Furthermore, activation of the enzyme occurred by directly adding serotonin or cyclic 3',5'-AMP to cell-free extracts of the flukes. Since serotonin specifically increases the synthesis of cyclic 3'-5'-AMP in the flukes (6), the hypothesis was put forward that serotonin activates the enzyme through the cyclic adenylic nucleotide. Following these findings on the flukes we became interested in understanding the different mechanisms involved in the regulation of this enzyme. I shall attempt to describe here some of the experiments we have carried out on the enzyme from two sources. The first is the liver fluke enzyme which has been partially purified and the second is the enzyme from the sheep heart which has been purified to homogeneity and crystallized (7). MATERIALS AND METHODS Cellular fractions from homogenates of the flukes were prepared by differential centrifugation and assays were carried out as described before (8, 9). Phosphofructokinase from the sheep heart was purified and crystallized 37
38
TAG E. MANSOUR
by our reported procedure (7). Enzyme activity was determined spectrophotometrically with coupled-enzyme systems (10, 11). Photo-oxidation of the enzyme in the presence of methylene blue was performed according to the method described by Ahlfors and Mansour (12). Enzyme has been referred to as desensitized or photo-oxidized enzyme if it was no longer inhibited by ATP at pH 6.9. The binding properties of phosphofructokinase were determined using the method of gel filtration described by Hummel and Dreyer (13). Other procedures for measuring binding of different ligands specific for phosphofructokinase was reported by Lorenson and Mansour (14).
RESULTS AND DISCUSSION
Evidence for an Inactit, e-Actit~e Conversion of Phosphofructinokinase A. Liverfluke enzyme. Evidence for the presence of inactive-active type of conversion for phosphofructokinase similar to that known for glycogen phosphorylase comes primarily from studies on the liver fluke enzyme. Following the demonstration that both serotonin and cyclic 3 ' , 5 ' - A M P (4, 5) activate phosphofructokinase attempts were made to explain such activation (8, 9). Fractionation of fluke homogenates revealed that an inactive form of the enzyme was present in cell fractions isolated between 80,000 and 150,000 x g centrifugation (80-150 fraction). Activation of this form of the enzyme by serotonin or cyclic 3 ' , 5 ' - A M P can be achieved only in the presence of a particulate fraction (80,000 x g), ATP and Mg + ' (Table 1). There is a distinct difference between activation of the enzyme in this system by the cyclic nucleotide and by serotonin. Incubation of the particulate fraction with ATP and Mg ++ produces a thermostabile factor(s) that activates the inactive
TABLE 1 EFFECT OF SEROTONIN AND OF CYCLtC 3 ' , 5 ' - A M P ON LIVER FLUKE PHOSPHOFRUCTOKINASE IN THE PRESENCE OF DIFFERENT CELL FRACTIONS
Cell fractions from homogenates of liver flukes were prepared as described before (8). Phosphofructokinase activity of all fractions is expressed as units per gram of flukes (wet weight). Data are from that of Stone and Mansour (8). Phosphofructokinase activity Cell fraction Homogenate (80-150)-Sediment* 80-Sedimentt (80-150)-Sediment + 80-Sediment
Control + Serotonin 10-4 M q-CyclicY,5"-AMP10-~ 0.38 0 0.11 0.63
1.53 0 0.15 1.69
1.91 0 0.2
1.87
*Cell fraction isolated between 80,000 x g and 150,000 x g centrifugation. tCell fraction isolated after 80,000 x g centrifugation.
CONTROL OF PHOSPHOFRUCTOKINASE
39
enzyme in the presence of cyclic 3',5'-AMP (at concentrations as low as l0 -s M) (Table 2). An absolute requirement for cyclic-3 ',5'-AMP is established TABLE 2
ACTIVATION OF PHOSPHOFRUCTOKINASE FROM THE LIVER FLUKE
Sediment containing inactive phosphofructokinase (80-150 sediment) was incubated with the indicated additions. Thermostable fraction A was prepared by incubating the 80,000 x g sediment with ATP and Mg ++. Thermostable fraction B was prepared under the same conditions except serotonin 10-' Mand caffeine 1.3 × l0 -2 u were included. After incubation for 12 rain at 30°, the mixture was diluted 200-fold and assayed. For further detail see reference (8). Phosphofructokinase activity (units/ml 80-150 sediment)
Exp. No.
Additions
1
None Cyclic 3",5"-AMP Thermostable fraction A Thermostabl¢ fraction A 4- 10-4 Mcyclic Y,5'-AMP
0.3 0.23 0.41
None 10-4 MSerotonin 4- 1.3 × 10-2 Mcaffeine + Thermostable fraction B
0.2 0.2 1.2
2.15
since no other nucleotide could replace it. On the other hand, incubation of the particulate fraction with ATP, Mg ++, serotonin and caffeine produces a thermostable factor(s) that activates the enzyme in the absence of cyclic 3 ' , 5 ' - A M P (Table 2). This finding supports our previously held view that the effect of serotonin is mediated through increasing the production of cyclic 3',5'-AMP. Analysis of the factors produced by incubating the particulate fraction with ATP and Mg ++ revealed that it contained considerable amounts of ATP, ADP, A M P and inorganic phosphate. A synthetic mixture of the same composition as the adenine nucleotides and inorganic phosphate activated the enzyme in the presence of cyclic 3',5'-AMP and Mg +÷. Systematic omission of components from the synthetic mixture indicated that in addition to cyclic 3',5'-AMP and Mg +÷, inorganic phosphate and A D P are necessary for optimal activation. The process of enzyme activation appears to be reversible since the activated enzyme can readily be inactivated by dialysis. The dialyzed inactivated enzyme can then be reactivated. Further evidence that such enzyme activation involves a conversion of inactive to active enzyme comes from measurement of sedimentation velocities of both forms of the enzyme. The inactive enzyme sediments with an $2~ value of 5.5 while active enzyme sediments at 12.8 S. This indicates that activation of the fluke phosphofructokinase involves polymerization of the enzyme. Further details of the system for activation and inactivation awaits enzyme purification.
40
TAG E. MANSOUR
B. Sheep heart enzyme in crude homogenate. It was first observed (10) that heart homogenates of freshly sacrificed guinea pigs contain a fully active phosphofructokinase which is in a soluble fraction (24,000 x g). Similar experiments with the sheep heart homogenate (7) revealed no significant activity in the homogenate nor in the soluble fraction (24,000 x g). However, when the residue of the sheep heart was incubated at 37 ° for 20 min with ATP and MgSO 4, high phosphofructokinase activity was observed in the supernatant fluid of the incubate (24,000 × g) (Table 3). The enzyme in this fracTABLE 3 EXTRACTION OF PHOSPHOFRUCTOKINASE FROM MAMMALIANHEARTS Homogenates were prepared by homogenizing 1 g of tissue in 4 ml of 0.01 M tris-Cl buffer (pH 8.0) in 0.002 M EDTA. Residue was isolated after centrifugation at 24,000 × g for 20 min. This supernatant fluid is "Supernatant l". The residue was then suspended in the same homogenizing solution, sedimented again by centrifugation and then resuspended in a solution containing 0.05 M MgSOo 0.005 M mereaptoethanol, 0.01 M tris-Cl (pH 8.0) and 5 x 10-4 M ATP. The suspension was stirred in a bath at 37 ° for 20 min and then centrifuged at 24,000 × g for 30 min at 0 °. The supematant fluid of this incubate is referred to as "Supernatant II". Fresh hearts from guinea pigs were prepared immediately after the animals were sacrificed. Aged heart homogenates were prepared from animals left half an hour after death at room temperature. The hearts were placed in ice for 45 rain and then homogenized. Phosphofructokinase activity (units/g wet weight) Exp.
Heart
Homogenate
Supernatant I
Residue
Supernatant II
2.9
0.1
0.3
21.9
Fresh G.P.* Aged G.P.
31.8 14.5
37.2 16.1
3.8 3.9
0.8 5.6
Fresh G.P. Aged G.P.
23.0 8.4
33.0 14.7
1.0 0.5
0.7 5.3
Fresh G.P. Aged G.P.
29.0 14.7
27.5 10.3
NDt ND
2.0 7.8
Sheep
*G.P. is guinea pig. 1'Not determined.
tion had a high specific activity and was used as a starting material for its purification (7). Hearts from the cow and pig which were collected from the slaughter house showed the same properties described for sheep heart enzyme. Experiments on the conditions necessary for enzyme activation and solubilization showed that AMP, ADP or cyclic 3',5'-AMP can replace ATP in the activating mixture. MgSO4 at a concentration of 0.1 M in the absence of ATP was also effective. This property of the enzyme appears to be restricted only
CONTROL OF PHOSPHOFRUCTOKINASE
41
to phosphofructokinase in muscle tissues of the sheep while in the brain, kidney, and liver phosphofructokinase remains soluble and active without such treatment. The question then arose: why is the guinea pig enzyme fully active and soluble while the sheep enzyme is inactive and can be spun down as part of the particulate fraction? An important factor which was considered was the fact that the homogenates from the guinea pig were prepared from freshly isolated hearts while those from the sheep were, by necessity, not fresh because of the time taken for transportation from the slaughter house to the laboratory. It then seemed likely that aging of the heart might result in having the guinea pig enzyme in a form similar to that of the sheep. Experiments summarized in Table 3 show that homogenates from guinea pigs which were left to age for ~ hr after killing had less than half of the enzyme activity in homogenates from freshly killed guinea pigs. Furthermore, residues from hearts of aged animals, when incubated with ATP and MgSO4 yielded activated soluble phosphofructokinase (Table 3). On the other hand, residues from hearts of freshly killed guinea pigs incubated in the same mixture did not yield significant enzyme activity. Experiments also showed that aging the homogenates of freshly isolated hearts does not have the same effect. The results suggest a possible conversion of the enzyme from active to inactive form during aging of the guinea pig heart.
C. Reversible dissociation of crystalline heart phosphofructokinase to subactive subunits. Experiments on both partially purified heart enzyme (15) and purified crystalline enzyme (7) revealed that they are capable of being reversibly converted to a form which is almost inactive. At pH 5.8 to 6.5 the enzyme is extremely unstable and when present in low concentrations it irreversibly loses its activity. On the other hand, at moderately high protein concentrations the inactivated enzyme can be reactivated by incubation at pH 8.0. Reactivation appears to be dependent on enzyme concentration, suggesting a reaction order greater than one. Reactivation of the enzyme is increased upon addition of ATP, ADP, cyclic 3',5'-AMP, fructose-6-P or fructose-l,6di-P. Combinations of hexose phosphates and one of the adenine nucleotides are much more effective than either nucleotide or hexose phosphate alone. The sedimentation velocity of the inactive phosphofructokinase was found to vary from 7.0 S to 8.0 S while the native fully active enzyme has a minimum S~ow value of 14.5 (Fig. 1). When the enzyme from the 7 S fraction was reactivated it sedimented like the native enzyme. Sedimentation velocity studies on the enzyme at high concentrations demonstrated that the enzyme has a high tendency to aggregate. At concentrations above 1 mg/ml the sedimentation coefficient varied from 15 S to 50 S. The relationship between the degree of aggregation of the enzyme and its catalytic activity cannot be ascertained because enzyme concentration during
42
TAG E. MANSOUR 6000
l
I
$2'0w=7.5
I
szo?,:,4.,
~ooo•= 4000 -
ion
g
c ~ooo-
2000 -
,,c
t befor~
|
.CI~Q.
me 5
10
~ T t5 Fraction
20
25
number
FIG. l Sedimentation patterns o f phosphofructokinase in sucrose gradient. The ermyme was dialyzed overnight against a solution containing 0.001 M dithioerythritol, 0.001 M E D T A and 0.2 M potassium phosphate buffer (pH 8.0 or p H 6.5). Concentration o f sucrose in the gradient varied from 3 9/00 (right side) to 1 0 ~ . Curves for p H 6.5 -- et-t-t-t-t-t-t-t-t-t~wme are shown before activation ( { ) - - t D ) and after activation of the enzyme fractions ( 0 - - 0 ) . F r o m M a n s o u r and Ahlfors (7).
its assay was always less than 0.05 tzg/ml, Since the degree of aggregation is concentration dependent, the enzyme at assay levels is presumably in the dissociated form.
Allosteric Kinetics of Phosphofructokinase Lardy and Parks (16) reported that ATP in concentrations above the molar equivalent of Mg ++ inhibited the activity of phosphofructokinase from the skeletal muscle. Subsequently a similar inhibitory effect was observed with phosphofructokinase from many other sources including the activated enzyme from the liver fluke (5). Saturation curve for fructose-6-P with the fluke enzyme was shown to be sigmoidal. Cyclic 3',5'-AMP reduced the apparent Km for fructose-6-P and shifted the sigmoidal kinetics to hyperbolic kinetics. The cyclic nucleotide was also shown to cause an increase in Ks for ATP. Recent studies on the fluke enzyme were undertaken in order to better understand the nature of interaction between different ligands and the protein. These experiments were carried out on the enzyme after it was fully activated in the usual system containing cyclic 3',5'-AMP (9). At non-inhibitory concentrations of ATP the saturation curve for fructose-6-P was not a Michaelis-
CONTROL OF PHOSPHOFRUCTOKINASE
43
Menton hyperbola, but rather it was sigmoidal in nature. Hill plots for this saturation curve gave a Hill coefficient greater than one indicating cooperative substrate binding. Addition of cyclic 3'-5'-AMP increases the apparent affinity of the enzyme for fructose-6-P and decreases the Hill coefficient indicating a decrease in cooperativity of fructose-6-P binding. The apparent Vmaz was not affected to a significant degree. Increasing ATP concentration increased the sigmoidicity of the fructose-6-P saturation curve. Similarly, increasing fructose-6-P concentration increases the apparent K~ for ATP without affecting the apparent Kin. Furthermore, increasing the concentration of fructose-6-P increased the apparent affinity for cyclic 3',5'-AMP, while increasing the ATP concentration had the opposite effect. It should be mentioned here that while 5-AMP cannot substitute for cyclic 3',5'-AMP in activation of the inactive enzyme of the liver fluke, it does have the same kinetic effects as the cyclic adenylic nucleotide. Interaction among the substrates and the effector ligands on the enzyme can be explained in terms of the Monod-Changeux-Wyman model (17). Subsequent to the kinetic effects we reported with the fluke enzyme (5), several enzymes have been studied with strikingly similar results (10, 18-24). Kinetic studies with the heart enzyme (10, 24) in our laboratory revealed that at pH 8.2 heart phosphofructokinase exhibits Michaelis-Menton kinetics with respect to ATP and to fructose-6-P. At pH 6.9, on the other hand, there is a sigmoidal kinetics to one of the substrates, fructose-6-P. Under the same conditions and with suboptimal concentration of fructose-6-P the enzyme is inhibited by ATP, citrate and creatine-P (25, 26). The enzyme, once inhibited, can be activated (or deinhibited) by fructose-6-P, fructose-l,6-di-P, AMP, cyclic 3',5'-AMP and P,. Kinetic data suggested that the ability of these activators to bvercome ATP inhibition is through an effect at an ATP site other than the catalytic site. While the apparent Km for ATP is not significantly changed by these activators, the apparent K, is increased. As in the case with the fluke enzyme, the activators affect the heart enzyme by decreasing the apparent Km for fructose-6-P. The multimolecular nature of the kinetics of the mammalian enzyme can also be explained on the basis of the M o n o d Changeux-Wyman model (! 7). Analysis of the kinetics by means of the Hill equation as proposed by Monod et aL reveals that the maximum apparent order of the reaction with respect to fructose-6-P is as high as 3.4. Different activators, such as 5-AMP, cyclic 3',5'-AMP and ADP, reduced the order of the reaction, or in the terminology of Monod et al. decreased the apparent cooperative interaction. The order of the reaction was found to be a function of the ATP concentration.
Modification of the Kinetics of Phosphofructokinase by Sulfhydryl Agents Thiol group analysis showed that phosphofructokinase from sheep heart contains approximately 56-58 sulfhydryl groups per 400,000 g and no
44
TAG E. MANSOUR
disulfide b o n d s (27). This n u m b e r is close to w h a t was r e p o r t e d b y Y o u n a t h a n , P a e t k a u a n d L a r d y (28) for the r a b b i t muscle enzyme. O f these thiol groups, only 25 are accessible by sulfhydryl agents in the native enzyme (in the absence o f 5 M urea). Evidence that the nature o f these g r o u p s are not identical comes f r o m the following o b s e r v a t i o n s : (a) reaction o f the enzyme with cysteine results in a biphasic inactivation curve; (b) titration with p c h l o r o m e r c u r i c b e n z o a t e ( p - C M B ) showed that the first 12 s u l f h y d r y l g r o u p s titrate at the s a m e rate in the presence o r absence o f A T P or fructose-6-P. However, titration o f the second 12 g r o u p s is r e d u c e d in the presence o f A T P or fructose-6-P. T h e effect o f titrating the thiol g r o u p s on enzyme activity was d e p e n d e n t on the s u l f h y d r y l reagent used a n d on pH. I n general, these agents inhibited enzyme activity m o r e at an acidic p H t h a n at an alkaline pH. T h e disulfide reagents were the m o s t p o t e n t inhibitors while the m o n o v a l e n t mercurials were the least inhibitory. T h e role o f thiol g r o u p s on the kinetics o f p h o s p h o fructokinase was investigated. This was carried out by titration o f a p p r o x i mately 15 thiol g r o u p s per 400,000 g o f enzyme with p - C M B . U n d e r these c o n d i t i o n s a n d at p H 8.2 the Vrnax for fructose-6-P was not c h a n g e d while the Km was increased by a factor o f 5. O n the o t h e r hand, A T P titration curves showed no c h a n g e in Km b u t a significant decrease in Vmax. T h e effect o f such t i t r a t i o n on the kinetics at p H 6.9 was m o r e significant. These results are s u m m a r i z e d in T a b l e 4. T h e most interesting effect which can be seen was a decrease in c o o p e r a t i v i t y o f the enzyme as d e t e r m i n e d by the Hill coefficient. I n the absence o f A M P there is a decrease in the Hill coefficient from 3.4 to 2.2 as a result o f the sulfhydryl g r o u p titration. W h i l e in the presence o f the TABLE4 p]-] 6.7 BY p-CHLOROMERCUR1C BENZOATE(p-CMB) Enzyme was allowed to react with p-CMB (15 thiol groups per 400,000 g of enzyme). Activity was determined in the presence of I mM MgCI~ and 0.16 mM ATP. The concentration of AMP when present was 0.1 mM. Reaction was started by adding ATP. From the data of Froede and Mansour (27). M O D I F I C A T I O N OF K I N E T I C S OF P H O S P H O F R U U ' r O K I N A S E A T
gm Hill V max 5-AMP p-CMB (/~mole fructose-i,6-di-P/mg/min) (Fructose-6-P 10-*M) coefficient* -
--
+
--
--
+
+
--
55 48 19 32
3.8 0.8 4.2 1.0
3.4 2.0 2.2 1.0
*Hill coefficient was determined from the following equation: v log Vmax - v n logS ~ logK where S is substrate concentration, K is constant, v is observed activity, and Vrnaxis maximum activity extrapolated from a Lineweaver-Burk plot of the rates.
CONTROL OF PHOSPHOFRUCTOKINASE
4.5
adenylic nucleotide (or cyclic 3',5'-AMP) the Hill coefficient was decreased from 2.0 to 1.0. Thus positive effectors such as 5-AMP or cyclic 3',5'-AMP were capable of changing the cooperative kinetics ofp-CMB-treated enzyme to first order kinetics. This would indicate that the interaction between the fructose-6-P binding sites could be uncoupled when these thiol groups are titrated. In contrast, neither positive effector alone could completely convert the cooperative kinetics of the native enzyme to first order kinetics. The effects of sulfhydryl agents on the kinetics of phosphofructokinase described above do not appear to be due to the presence of these agents at the active site. This is supported by the fact that neither substrate protected these thiol groups against titration with sulfhydryl agents. Furthermore, when all the thiol groups were titrated with p-CMB, enzyme activity at pH 8.2 was only reduced to 50% of the original activity. The changes in the catalytic site, therefore, could be induced through conformational change in the protein. The role of sulfhydryl groups in determining the quaternary structure of phosphofructokinase is not unique. The cooperative binding of oxygen by hemoglobin is changed by modifying the sulfhydryl group at position 93 of the B-chain (29-31). In aspartate transcarbamylase enzyme activity is increased and it loses its cooperative properties when treated with divalent and monovalent mercurials (32). To|
t~
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,
,
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I
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,
l\
°°Vg \ ,~
.~0~
o.o ~ o;
~ ~ 0 l ~
.;o
.;,
~o ~
i o
[ATP] (.raM)
~
f
F ~ .4,
.~o
FxG. 2 Plots of initial reaction velocity with respect to ATP concentration at pH 6.9 of native (O--O) and photo-oxidized enzyme (0--0). The insert shows a Lineweaver-Burkplot of the data. From Ahlfors and Mansour (12).
46
TAG E. MANSOUR
Desensitization of Sheep Heart Phosphofructokinase to Allosteric Control The discovery of the kinetics of phosphofructokinase introduced to the group of allosteric proteins a unique enzyme which has as an allosteric modifier one of its substrates. Both homotropic and heterotropic interactions are dependent on ATP concentrations. In an attempt to show conclusively that there are allosteric sites for ATP which are different from the catalytic sites, a modified form of the enzyme has recently been prepared by photo-oxidation in the presence of methylene blue (12). The photo-oxidized enzyme lost completely both its homotropic and heterotropic interaction. Neither ATP (Fig. 2)
-z
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/
t
t .^ I "
, ~ 0 201~ I 0 .5
i
.1::,
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Lo9 ~[~Cruc,o~-6 r P]x 104 ' I l' I I 10 1.5 2.0 2.5 3.0 ~,5 Irruc*ose -6 -P (m M) FiG. 3
Plots o f initial reaction velocity with respect to fructose-6-P at p H 6.9 of native ( O - - O ) and photo-oxidized ( O " - - Q ) enzyme. The insert shows plots derived from the Hill equation. F r o m Ahlfors and Mansour (12).
nor citrate could inhibit the enzyme. Furthermore, the Hill coefficient for fructose-6-P which was 3.15 for the native enzyme was reduced to 1.1 after photo-oxidation (Fig. 3). None of the activators (deinhibitors) had an effect on the modified enzyme. The Km for fructose-6-P at pH 6.9 was markedly reduced. All these effects were observed with only a slight reduction to the maximal enzyme activity. It therefore appears that photo-oxidation of the enzyme induces a specific conformation for the protein. This form of the enzyme shows no allosteric interaction, but the maximal activity is slightly affected. The question whether this form does not bind ATP at the allosteric sites is answered in the experiments described below.
47
CONTROL OF PHOSPHOFRUCTOKINASE
Binding Properties of Heart Phosphofructokinase Further evidence for the presence of allosteric sites which are different from the catalytic sites comes from recent studies on the binding properties of native and photo-oxidized enzyme (14). Binding studies were carried out using gel filtration technique (13). Some of these binding experiments are summarized in Table 5. While the native enzyme binds 14.4 moles of ATP per 400,000 g, TABLE 5 BINDING PROPERTIESOF NATIVE ENZYME AND OF ENZYME DESENSITIZEDTO ALLOSTERIC CONTROL BY PHOTO-OXIDATION Binding properties of phosphofructokinase were determined using the method of gel filtration described by Hummel and Dreyer (13). Data were calculated from doublereciprocal plots of substrate bound vs. free concentration of substrate. The dissociation constant (Ka) and the maximum theoretical number of binding sites per mole of protein (n) were determined by extrapolation of the reciprocal graphs. Lorenson and Mansour (14). Native Substrate ATP Citrate Fructose-6-P
Photo-oxidized
n
Ka
n
Ka
Mole/400,000 g 14.4 8.0 7.0
M 2 × l0-6 3 × l0 -r 2.1 × 10 -5
Mole/400,000 g 7.2 No binding 6.0
M 2.8 × 10-e No binding 2.0 × 10 -5
the photo-oxidized enzyme binds only 7.2 moles per 400,000 g. Citrate, which is not a substrate, but only an allosteric modifier binds to the native enzyme only to the extent of 8 moles per 400,000 g. On the other hand, the photooxidized enzyme does not bind any citrate. The binding of fructose-6-P was not affected by photo-oxidation. Both forms of the enzyme bind 7 moles of this substrate per 400,000 g. A conclusion can be drawn from this data that the native enzyme has 7 active sites and 7 allosteric sites. Both citrate and ATP bind at the same allosteric sites. Experiments failed to demonstrate a cooperative effect on the binding of fructose-6-P. This is possibly due to the fact that inorganic phosphate had to be used in these experiments in order to avoid non-specific binding. Under these conditions the kinetics for fructose-6-P are first order. Another possible reason for not demonstrating a cooperative effect is the fact that these studies were carried out in the absence of ATP to avoid starting enzyme reaction. The effect of pH on the binding of different ligands revealed some interesting findings which are summarized in Table 6. At a non-saturating ATP concentration of 10/zM the binding was markedly decreased at an alkaline pH. Furthermore, the enzyme did not bind citrate at an alkaline pH. The binding of fructose-6-P was not affected by pH. These experiments indicate that the binding to the allosteric sites is maximal at an acid pH. The results might explain the dependence ofallosteric interaction of the enzyme on an acidic pH.
48
TAG E. MANSOUR TABLE6 EFFECTOF pH ON THEBINDINGOF DIFFERENTSUBSTRATESBY PHOSPHOFRUCTOKINASE Binding of different substrates was determined at the indicated concentrations. From the data of Mansour and Lorenson (14). Substrate binding (molesper 400,000g) Substrate ATP 10/.zM ATP 10 t~M Citrate 5 /~M Citrate 5 ~M Fructose-6-P* Fructose-6-P*
pH
Native PFK
6 8 6 8 6.7 8.0
12.8 4.0 3.6 No binding 7.0 7.0
Photo-oxidized PFK 4
2 No binding No binding 6.4 6.4
*Maximal binding at infinite concentration of fructose-6-P was by extrapolation from reciprocal plots as described in Table 5.
In their studies on the binding properties of skeletal muscle phosphofructokinase Kemp and Krebs (33) reported that AMP, ADP and cyclic Y,5'-AMP bind at one common site per protomer (90,000 g). ATP can bind to the same site but it has a higher dissociation constant than that of the other nucleotides. The effect of the nucleotide activators, therefore, could be related to their competition with ATP for binding at this site. PHYSIOLOGICAL INTERPRETATIONS The above described experiments with both mammalian and fluke phosphofructokinases point to two main mechanisms which could be of importance from the standpoint of physiological regulation: I. Phosphofructokinase could be present in two forms, active and subactive. These two forms are interconvertible under certain conditions by an association-dissociation process. 2. Phosphofructokinase at pH 6.9 is allosterically inhibited by ATP, citrate and creatine phosphate. The inhibited enzyme can be activated by 5-AMP, cyclic 3',5'-AMP, A D P and P~. There is considerable evidence to support the conclusion that both mechanisms could be present in the liver fluke. The enzyme in the resting flukes is simply inactive, but is activated by serotonin. The activation is concomitant with an increase in glycolysis. The amine activates the enzyme and glycolysis in the cell-free extracts as well as in the intact organisms. The evidence here appears to be as strong as that available on the effect of epinephrine on mammalian liver phosphorylase. There is also considerable evidence that activation of the fluke enzyme is mediated through cyclic 3',5'-AMP although the details of the activating system have not yet been worked out because of
CONTROLOF PHOSPHOFRUCTOKINASE
49
the unavailability of a purified fluke enzyme. The fluke enzyme, once activated, is probably subject to allosteric control as described above. In the mammalian enzyme, although it can be reversibly converted to active and subactive forms, the evidence for the physiological importance of this process is incomplete. Freshly isolated tissues have always yielded a fully active enzyme. Our experience with these tissues is that they all show all the signs associated with anoxia. This simple fact throws some doubt on the assumption that enzyme levels of these tissues represent resting levels. The possibility, therefore, exists that in mammals, where the demand for oxidative metabolism is great, the enzyme is activated as soon as the tissues are removed from the animal. The role of phosphofructokinase in relation to anoxia has been postulated 0-3, 18). Evidence that the mammalian enzyme is regulated through its complex kinetic properties described above is indirect. An increase in glycolysis during anoxia is associated with a decrease in the intracellular levels of free glucose, glucose-6-P and fructose-6-P and a simultaneous increase in the concentration of fructose-l,6-di-P and the later glycolytic metabolites. These events coincide with an increase in the levels of 5'-AMP, ADP and Pt. An increase in the level of these agents could activate the enzyme by overcoming ATP inhibition of phosphofructokinase. Kinetic data suggest that phosphofructokinase can respond to what has been termed by Atkinson "'adenylate charge" (34, 35). Kinetic results reported by him with reactants and nucleotides at physiological concentrations showed that the enzyme could respond to quite small concentrations of AMP. A change in the adenylate charge from 1.0 to 0.9 results in an insignificant change in ATP concentration (negative modifier) but in a significant change in AMP levels (with a 4 mM adenylate pool the concentration of AMP is 0 at a charge of 1.0 and 100 tzM at a charge of 0.9). Citrate was shown to modulate this effect by increasing the steepness of the response curve to changes in the adenylate charge. Cyclic 3',5'-AMP acts directly on phosphofructokinase by releasing the enzyme from control through the adenylate system. This is compatible with our early observations on the fluke and the heart enzymes that the cyclic nucleotide renders the phosphofructokinase less susceptible to inhibition by ATP (5, I0). As it was pointed out by Atkinson (34) this hypothesis is subject to the limitation that we have no idea about the functional compartmentation in the living cell. Determinations of the levels of these substrates is always based on the assumption of their homogenous distribution within the cell. SUMMARY Kinetic and physical properties of phosphofructokinase from the liver fluke
Fasciola hepatica and from the mammalian heart were described. Evidence for the presence of an inactive-active conversion of the enzyme was presented.
50
TAG E. MANSOUR
T h e kinetic b e h a v i o r o f the enzyme was discussed in terms o f the M o n o d C h a n g e u x - W y m a n m o d e l for allosteric proteins. Studies were described elucidating the role o f thiol groups o n allosteric kinetics o f the enzyme. Evidence was presented showing that the allosteric site on the enzyme is different from the catalytic site. Binding properties o f the two sites to substrates a n d modifiers were presented. Possible physiological significance seen in all these properties was discussed in terms o f the regulation o f enzyme activity within the cell.
ACKNOWLEDGMENTS This research was s u p p o r t e d by U n i t e d States Public H e a l t h Service Research G r a n t A I O 4 2 1 4 from the N a t i o n a l Institute o f Allergy a n d Infectious Diseases; U n i t e d States Public H e a l t h Service C a r e e r D e v e l o p m e n t A w a r d G M 3848 f r o m the Division o f G e n e r a l M e d i c a l Sciences; a n d a G r a n t - i n - A i d from the A m e r i c a n H e a r t Association.
REFERENCES 1. Co F. CoRt, Phosphorylation of carbohydrates, pp. 175-189 in A Symposium on Respiratory Enzymes, University of Wisconsin Press, Madison (1942). 2. V. A. ENGEL'HARDTand N. E. SAKOV,The mechanism of the Pasteur effect, Biokhimiya 8, 9-36 (1943). 3. F. LYNEN, G. HARTMANN, K. F. NETTER and A. SCttUEGRAT, pp. 256-273 in Ciba Foundation Symposium on Regulation of Cell Metabolism (G. E. W. WOLSTENHOLME,ed.),
Little, Brown & Co., Boston (1959). 4. T. E. MANSOOR,Effect of serotonin on glycolysis in homogenates from the liver fluke, Fasciola hepatica, J. Pharmacol. Exptl. Therap. 135, 94-101 (1962). 5. T. E. MANSOURand J. M. MANSOUR, Effects of serotonin (5-hydroxytryptamine) and adenosine 3',5'-phosphate on phosphofructokinase from the liver fluke, Faseiola hepatica, J. Biol. Chem. 237, 629-634 (1962). 6. T. E. MANSOUR, E, W. SUTHERLAND,T. W. PALL and E. BUEDING, The effect of serotonin (5-hydroxytryptamine) on the formation of adenosine-Y,5'-phosphate by tissue particles from the liver fluke, Fasciola hepatica, J. Biol. Chem. 235, 466-470 (1960). 7. T. E. MANSOUR, N. WAKID and H. M. SPROUSE, Studies on heart phosphofructokinase: purification, crystallization and properties of sheep heart phosphofructokinase, J. BioL Chem. 241, 1512-1521 (1966). 8. D. B. STONEand T. E. MANSOUR,Phosphofruetokinase from the liver fluke, Fasciola hepatica. I. Activation by adenosine Y,5'-phosphate and by serotonin, Mol. Pharmacol.
3, 161-176 (1967). 9. D. B. STONEand T. E. MANSOUR,Phosphofructokinase from the liver fluke, Fasciola hepatica. II. Kinetic properties of the enzyme, Mol. Pharmacol. 3, 177-187 (1967). 10. T. E. MANSOUR,Studies on heart phosphofructokinase: purification, inhibition and activation, J. Biol. Chem. 238, 2285-2292 (1963). 11. T. E. MANSOUR, Phosphofructokinase II. Heart muscle, pp. 430-436 in Methods in Enzymology, Vol. IX (W. A. WOODS,ed.), Academic Press, Inc., New York 0968). 12. C. E. ArlLroRs and T. E. MANSOUR, Studies on heart phosphofructokinase: desensitization of the enzyme to adenosine triphosphate inhibition, J. Biol. Chem. 244, 1247-1251 (1969).
CONTROL OF PHOSPHOFRUCTOKINASE
51
13. J. P. HUMMELand W. J. D~vr.n, Measurement of proteln-binding phenomena by gel filtration, Biochim. Biophys. Acta 63, 530-532 (1962). 14. M. Y. LORENSONand T. E. MANSOLrP.,Studies on Heart phosphofructokinas¢: binding properties of native enzyme and of enzyme desensitized to allosteric control. J. Biol. Chem. 244, 6420-6431 (1969). 15. T. E. MANSOLrg,Studies on heart phosphofructokinas¢: active and inactive forms of the enzyme, J. Biol. Chem. 240, 2165-2172 (1965). 16. H. A. LARDYand R. E. PARKS, JR. Influence of ATP concentration on rates of some phosphorylation reactions, pp. 584-588 in Enzymes: Units of Biological Structure and Function (O. H. GAEnLER,ed.), Academic Press, Inc., New York (1956). 17. J. MONOD, J. WYMANand J.-P. CHANGEtrx, On the nature of allosteric transitions: a plausible model, J. Mol. Biol. 12, 88-118 (1965). 18. J. V. PASSOr,n~AU and O. H. LOWRY, Phosphofructokinase and the Pasteur effect, Biochem. Biophys. Res. Commun. 7, 10-15 (1962). 19. A. RAMAIAH,J. A. HATHAWAYand D. E. ATgrSSON, Adenylate as a metabolic regulator: effect on yeast phosphofructokinase kinetics, J. Biol. Chem. 239, 3619-3622 (1964). 20. A. PARM~t3GIA~ and R. H. BOWMAt~, Regulation of phosphofructokinas¢ activity by citrate in normal and diabetic muscle, Biochem. Biophys. Res. Commun. 12, 268-273 (1963). 21. P. B. GARLAND,P. J. RANDLEand E. A. NEWSHOLME,Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation, Nature 200, 169-170 (1963). 22. M. SALAS, E. Vn,rO~LA, M. SALASand A. Sot.s, Citrate inhibition of phosphofructokinase and the Pasteur effect, Biochem. Biophys. Res. Commun. 19, 371-376 (1965). 23. D. BLA~GY, H. Buc and J. MONOD, Kinetics of the allosteric interactions of phosphofructokinase from E. coli, J. Mol. Biol. 31, 13-35 (1968). 24. T. E. MANSOORand C. E. A~LFORS, Studies on heart phosphofructokinase: some kinetic and physical properties of the crystalline enzyme, J. Biol. Chem. 243, 2523-2533 (1968). 25. J. KRZANOWSKI and F. M. MA'I"SCI~NSKY, Regulation of phosphofructokinase by phosphocreatine and phosphorylated glycolytic intermediates, Biochem. Biophys. Res. Commun. 34, 816-823 (1969). 26. A. OTANI and T. E, MANSOUR,Unpublished observations. 27. H. C. FROF_DE,G. GERACÁand T. E. MANSOUR,Studies on heart phosphofructokinas~: thiol groups and their relationship to activity, d. Biol. Chem. 243, 6021-6029 (1968). 28. E. S. YOUNATHAN,V. PAETKAUand H. A. LARDY,Rabbit muscle phosphofructokinase reactivity and function of thiol groups, J. Biol. Chem. 243, 1603-1608 (1968). 29. A. R~G~S, The binding of N-ethylomaleimide by human hemoglobin and its effect upon the oxygen equilibrium, J. Biol. Chem. 236, 1948-1954 (1961). 30. R. BENESCHand R. E. BENESCH,The chemistry of the Bohr effect. I. The reaction of N-ethylmaleimide with the oxygen-linked acid groups of hemoglobin, J. Biol. Chem. 236, 405-410 (1961). 31. J. F. TAYLOn, E. Am~NI~n, M. Bgtmom and J. WYMAN,Studies on human hemoglobin treated with various sulfhydryl reagents, J. Biol. Chem. 241, 241-248 (1966). 32. J. C. G~a*.HARTand A. B. PARDEE,The enzymology of control by feedback inhibition, J. Biol. Chem. 237, 891-896 (1962). 33. R.. G. KEMP and E. G. Kilns, Binding of metabolites by phosphofructokinase, Biochemistry 6, 423-434 (1967). 34. D. E. ATKINSON, The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers, Biochemistry 7, 4030-4034 (1968). 35. L. C. SHEN,L. FALL,G. M. WALTONand D. E. ATKrNSON,Interaction between energy charge and metabolite modulation in the regulation of enzymes of amphibolic sequences. Phosphofructokinase and pyruvate dehydrogenas¢, Biochemistry 7, 4041--4045 (1968).
38
TAG E. MANSOUR
by our reported procedure (7). Enzyme activity was determined spectrophotometrically with coupled-enzyme systems (10, 11). Photo-oxidation of the enzyme in the presence of methylene blue was performed according to the method described by Ahlfors and Mansour (12). Enzyme has been referred to as desensitized or photo-oxidized enzyme if it was no longer inhibited by ATP at pH 6.9. The binding properties of phosphofructokinase were determined using the method of gel filtration described by Hummel and Dreyer (13). Other procedures for measuring binding of different ligands specific for phosphofructokinase was reported by Lorenson and Mansour (14).
RESULTS AND DISCUSSION
Evidence for an Inactit, e-Actit~e Conversion of Phosphofructinokinase A. Liverfluke enzyme. Evidence for the presence of inactive-active type of conversion for phosphofructokinase similar to that known for glycogen phosphorylase comes primarily from studies on the liver fluke enzyme. Following the demonstration that both serotonin and cyclic 3 ' , 5 ' - A M P (4, 5) activate phosphofructokinase attempts were made to explain such activation (8, 9). Fractionation of fluke homogenates revealed that an inactive form of the enzyme was present in cell fractions isolated between 80,000 and 150,000 x g centrifugation (80-150 fraction). Activation of this form of the enzyme by serotonin or cyclic 3 ' , 5 ' - A M P can be achieved only in the presence of a particulate fraction (80,000 x g), ATP and Mg + ' (Table 1). There is a distinct difference between activation of the enzyme in this system by the cyclic nucleotide and by serotonin. Incubation of the particulate fraction with ATP and Mg ++ produces a thermostabile factor(s) that activates the inactive
TABLE 1 EFFECT OF SEROTONIN AND OF CYCLtC 3 ' , 5 ' - A M P ON LIVER FLUKE PHOSPHOFRUCTOKINASE IN THE PRESENCE OF DIFFERENT CELL FRACTIONS
Cell fractions from homogenates of liver flukes were prepared as described before (8). Phosphofructokinase activity of all fractions is expressed as units per gram of flukes (wet weight). Data are from that of Stone and Mansour (8). Phosphofructokinase activity Cell fraction Homogenate (80-150)-Sediment* 80-Sedimentt (80-150)-Sediment + 80-Sediment
Control + Serotonin 10-4 M q-CyclicY,5"-AMP10-~ 0.38 0 0.11 0.63
1.53 0 0.15 1.69
1.91 0 0.2
1.87
*Cell fraction isolated between 80,000 x g and 150,000 x g centrifugation. tCell fraction isolated after 80,000 x g centrifugation.
CONTROL OF PHOSPHOFRUCTOKINASE
39
enzyme in the presence of cyclic 3',5'-AMP (at concentrations as low as l0 -s M) (Table 2). An absolute requirement for cyclic-3 ',5'-AMP is established TABLE 2
ACTIVATION OF PHOSPHOFRUCTOKINASE FROM THE LIVER FLUKE
Sediment containing inactive phosphofructokinase (80-150 sediment) was incubated with the indicated additions. Thermostable fraction A was prepared by incubating the 80,000 x g sediment with ATP and Mg ++. Thermostable fraction B was prepared under the same conditions except serotonin 10-' Mand caffeine 1.3 × l0 -2 u were included. After incubation for 12 rain at 30°, the mixture was diluted 200-fold and assayed. For further detail see reference (8). Phosphofructokinase activity (units/ml 80-150 sediment)
Exp. No.
Additions
1
None Cyclic 3",5"-AMP Thermostable fraction A Thermostabl¢ fraction A 4- 10-4 Mcyclic Y,5'-AMP
0.3 0.23 0.41
None 10-4 MSerotonin 4- 1.3 × 10-2 Mcaffeine + Thermostable fraction B
0.2 0.2 1.2
2.15
since no other nucleotide could replace it. On the other hand, incubation of the particulate fraction with ATP, Mg ++, serotonin and caffeine produces a thermostable factor(s) that activates the enzyme in the absence of cyclic 3 ' , 5 ' - A M P (Table 2). This finding supports our previously held view that the effect of serotonin is mediated through increasing the production of cyclic 3',5'-AMP. Analysis of the factors produced by incubating the particulate fraction with ATP and Mg ++ revealed that it contained considerable amounts of ATP, ADP, A M P and inorganic phosphate. A synthetic mixture of the same composition as the adenine nucleotides and inorganic phosphate activated the enzyme in the presence of cyclic 3',5'-AMP and Mg +÷. Systematic omission of components from the synthetic mixture indicated that in addition to cyclic 3',5'-AMP and Mg +÷, inorganic phosphate and A D P are necessary for optimal activation. The process of enzyme activation appears to be reversible since the activated enzyme can readily be inactivated by dialysis. The dialyzed inactivated enzyme can then be reactivated. Further evidence that such enzyme activation involves a conversion of inactive to active enzyme comes from measurement of sedimentation velocities of both forms of the enzyme. The inactive enzyme sediments with an $2~ value of 5.5 while active enzyme sediments at 12.8 S. This indicates that activation of the fluke phosphofructokinase involves polymerization of the enzyme. Further details of the system for activation and inactivation awaits enzyme purification.
40
TAG E. MANSOUR
B. Sheep heart enzyme in crude homogenate. It was first observed (10) that heart homogenates of freshly sacrificed guinea pigs contain a fully active phosphofructokinase which is in a soluble fraction (24,000 x g). Similar experiments with the sheep heart homogenate (7) revealed no significant activity in the homogenate nor in the soluble fraction (24,000 x g). However, when the residue of the sheep heart was incubated at 37 ° for 20 min with ATP and MgSO 4, high phosphofructokinase activity was observed in the supernatant fluid of the incubate (24,000 × g) (Table 3). The enzyme in this fracTABLE 3 EXTRACTION OF PHOSPHOFRUCTOKINASE FROM MAMMALIANHEARTS Homogenates were prepared by homogenizing 1 g of tissue in 4 ml of 0.01 M tris-Cl buffer (pH 8.0) in 0.002 M EDTA. Residue was isolated after centrifugation at 24,000 × g for 20 min. This supernatant fluid is "Supernatant l". The residue was then suspended in the same homogenizing solution, sedimented again by centrifugation and then resuspended in a solution containing 0.05 M MgSOo 0.005 M mereaptoethanol, 0.01 M tris-Cl (pH 8.0) and 5 x 10-4 M ATP. The suspension was stirred in a bath at 37 ° for 20 min and then centrifuged at 24,000 × g for 30 min at 0 °. The supematant fluid of this incubate is referred to as "Supernatant II". Fresh hearts from guinea pigs were prepared immediately after the animals were sacrificed. Aged heart homogenates were prepared from animals left half an hour after death at room temperature. The hearts were placed in ice for 45 rain and then homogenized. Phosphofructokinase activity (units/g wet weight) Exp.
Heart
Homogenate
Supernatant I
Residue
Supernatant II
2.9
0.1
0.3
21.9
Fresh G.P.* Aged G.P.
31.8 14.5
37.2 16.1
3.8 3.9
0.8 5.6
Fresh G.P. Aged G.P.
23.0 8.4
33.0 14.7
1.0 0.5
0.7 5.3
Fresh G.P. Aged G.P.
29.0 14.7
27.5 10.3
NDt ND
2.0 7.8
Sheep
*G.P. is guinea pig. 1'Not determined.
tion had a high specific activity and was used as a starting material for its purification (7). Hearts from the cow and pig which were collected from the slaughter house showed the same properties described for sheep heart enzyme. Experiments on the conditions necessary for enzyme activation and solubilization showed that AMP, ADP or cyclic 3',5'-AMP can replace ATP in the activating mixture. MgSO4 at a concentration of 0.1 M in the absence of ATP was also effective. This property of the enzyme appears to be restricted only
CONTROL OF PHOSPHOFRUCTOKINASE
41
to phosphofructokinase in muscle tissues of the sheep while in the brain, kidney, and liver phosphofructokinase remains soluble and active without such treatment. The question then arose: why is the guinea pig enzyme fully active and soluble while the sheep enzyme is inactive and can be spun down as part of the particulate fraction? An important factor which was considered was the fact that the homogenates from the guinea pig were prepared from freshly isolated hearts while those from the sheep were, by necessity, not fresh because of the time taken for transportation from the slaughter house to the laboratory. It then seemed likely that aging of the heart might result in having the guinea pig enzyme in a form similar to that of the sheep. Experiments summarized in Table 3 show that homogenates from guinea pigs which were left to age for ~ hr after killing had less than half of the enzyme activity in homogenates from freshly killed guinea pigs. Furthermore, residues from hearts of aged animals, when incubated with ATP and MgSO4 yielded activated soluble phosphofructokinase (Table 3). On the other hand, residues from hearts of freshly killed guinea pigs incubated in the same mixture did not yield significant enzyme activity. Experiments also showed that aging the homogenates of freshly isolated hearts does not have the same effect. The results suggest a possible conversion of the enzyme from active to inactive form during aging of the guinea pig heart.
C. Reversible dissociation of crystalline heart phosphofructokinase to subactive subunits. Experiments on both partially purified heart enzyme (15) and purified crystalline enzyme (7) revealed that they are capable of being reversibly converted to a form which is almost inactive. At pH 5.8 to 6.5 the enzyme is extremely unstable and when present in low concentrations it irreversibly loses its activity. On the other hand, at moderately high protein concentrations the inactivated enzyme can be reactivated by incubation at pH 8.0. Reactivation appears to be dependent on enzyme concentration, suggesting a reaction order greater than one. Reactivation of the enzyme is increased upon addition of ATP, ADP, cyclic 3',5'-AMP, fructose-6-P or fructose-l,6di-P. Combinations of hexose phosphates and one of the adenine nucleotides are much more effective than either nucleotide or hexose phosphate alone. The sedimentation velocity of the inactive phosphofructokinase was found to vary from 7.0 S to 8.0 S while the native fully active enzyme has a minimum S~ow value of 14.5 (Fig. 1). When the enzyme from the 7 S fraction was reactivated it sedimented like the native enzyme. Sedimentation velocity studies on the enzyme at high concentrations demonstrated that the enzyme has a high tendency to aggregate. At concentrations above 1 mg/ml the sedimentation coefficient varied from 15 S to 50 S. The relationship between the degree of aggregation of the enzyme and its catalytic activity cannot be ascertained because enzyme concentration during
42
TAG E. MANSOUR 6000
l
I
$2'0w=7.5
I
szo?,:,4.,
~ooo•= 4000 -
ion
g
c ~ooo-
2000 -
,,c
t befor~
|
.CI~Q.
me 5
10
~ T t5 Fraction
20
25
number
FIG. l Sedimentation patterns o f phosphofructokinase in sucrose gradient. The ermyme was dialyzed overnight against a solution containing 0.001 M dithioerythritol, 0.001 M E D T A and 0.2 M potassium phosphate buffer (pH 8.0 or p H 6.5). Concentration o f sucrose in the gradient varied from 3 9/00 (right side) to 1 0 ~ . Curves for p H 6.5 -- et-t-t-t-t-t-t-t-t-t~wme are shown before activation ( { ) - - t D ) and after activation of the enzyme fractions ( 0 - - 0 ) . F r o m M a n s o u r and Ahlfors (7).
its assay was always less than 0.05 tzg/ml, Since the degree of aggregation is concentration dependent, the enzyme at assay levels is presumably in the dissociated form.
Allosteric Kinetics of Phosphofructokinase Lardy and Parks (16) reported that ATP in concentrations above the molar equivalent of Mg ++ inhibited the activity of phosphofructokinase from the skeletal muscle. Subsequently a similar inhibitory effect was observed with phosphofructokinase from many other sources including the activated enzyme from the liver fluke (5). Saturation curve for fructose-6-P with the fluke enzyme was shown to be sigmoidal. Cyclic 3',5'-AMP reduced the apparent Km for fructose-6-P and shifted the sigmoidal kinetics to hyperbolic kinetics. The cyclic nucleotide was also shown to cause an increase in Ks for ATP. Recent studies on the fluke enzyme were undertaken in order to better understand the nature of interaction between different ligands and the protein. These experiments were carried out on the enzyme after it was fully activated in the usual system containing cyclic 3',5'-AMP (9). At non-inhibitory concentrations of ATP the saturation curve for fructose-6-P was not a Michaelis-
CONTROL OF PHOSPHOFRUCTOKINASE
43
Menton hyperbola, but rather it was sigmoidal in nature. Hill plots for this saturation curve gave a Hill coefficient greater than one indicating cooperative substrate binding. Addition of cyclic 3'-5'-AMP increases the apparent affinity of the enzyme for fructose-6-P and decreases the Hill coefficient indicating a decrease in cooperativity of fructose-6-P binding. The apparent Vmaz was not affected to a significant degree. Increasing ATP concentration increased the sigmoidicity of the fructose-6-P saturation curve. Similarly, increasing fructose-6-P concentration increases the apparent K~ for ATP without affecting the apparent Kin. Furthermore, increasing the concentration of fructose-6-P increased the apparent affinity for cyclic 3',5'-AMP, while increasing the ATP concentration had the opposite effect. It should be mentioned here that while 5-AMP cannot substitute for cyclic 3',5'-AMP in activation of the inactive enzyme of the liver fluke, it does have the same kinetic effects as the cyclic adenylic nucleotide. Interaction among the substrates and the effector ligands on the enzyme can be explained in terms of the Monod-Changeux-Wyman model (17). Subsequent to the kinetic effects we reported with the fluke enzyme (5), several enzymes have been studied with strikingly similar results (10, 18-24). Kinetic studies with the heart enzyme (10, 24) in our laboratory revealed that at pH 8.2 heart phosphofructokinase exhibits Michaelis-Menton kinetics with respect to ATP and to fructose-6-P. At pH 6.9, on the other hand, there is a sigmoidal kinetics to one of the substrates, fructose-6-P. Under the same conditions and with suboptimal concentration of fructose-6-P the enzyme is inhibited by ATP, citrate and creatine-P (25, 26). The enzyme, once inhibited, can be activated (or deinhibited) by fructose-6-P, fructose-l,6-di-P, AMP, cyclic 3',5'-AMP and P,. Kinetic data suggested that the ability of these activators to bvercome ATP inhibition is through an effect at an ATP site other than the catalytic site. While the apparent Km for ATP is not significantly changed by these activators, the apparent K, is increased. As in the case with the fluke enzyme, the activators affect the heart enzyme by decreasing the apparent Km for fructose-6-P. The multimolecular nature of the kinetics of the mammalian enzyme can also be explained on the basis of the M o n o d Changeux-Wyman model (! 7). Analysis of the kinetics by means of the Hill equation as proposed by Monod et aL reveals that the maximum apparent order of the reaction with respect to fructose-6-P is as high as 3.4. Different activators, such as 5-AMP, cyclic 3',5'-AMP and ADP, reduced the order of the reaction, or in the terminology of Monod et al. decreased the apparent cooperative interaction. The order of the reaction was found to be a function of the ATP concentration.
Modification of the Kinetics of Phosphofructokinase by Sulfhydryl Agents Thiol group analysis showed that phosphofructokinase from sheep heart contains approximately 56-58 sulfhydryl groups per 400,000 g and no
44
TAG E. MANSOUR
disulfide b o n d s (27). This n u m b e r is close to w h a t was r e p o r t e d b y Y o u n a t h a n , P a e t k a u a n d L a r d y (28) for the r a b b i t muscle enzyme. O f these thiol groups, only 25 are accessible by sulfhydryl agents in the native enzyme (in the absence o f 5 M urea). Evidence that the nature o f these g r o u p s are not identical comes f r o m the following o b s e r v a t i o n s : (a) reaction o f the enzyme with cysteine results in a biphasic inactivation curve; (b) titration with p c h l o r o m e r c u r i c b e n z o a t e ( p - C M B ) showed that the first 12 s u l f h y d r y l g r o u p s titrate at the s a m e rate in the presence o r absence o f A T P or fructose-6-P. However, titration o f the second 12 g r o u p s is r e d u c e d in the presence o f A T P or fructose-6-P. T h e effect o f titrating the thiol g r o u p s on enzyme activity was d e p e n d e n t on the s u l f h y d r y l reagent used a n d on pH. I n general, these agents inhibited enzyme activity m o r e at an acidic p H t h a n at an alkaline pH. T h e disulfide reagents were the m o s t p o t e n t inhibitors while the m o n o v a l e n t mercurials were the least inhibitory. T h e role o f thiol g r o u p s on the kinetics o f p h o s p h o fructokinase was investigated. This was carried out by titration o f a p p r o x i mately 15 thiol g r o u p s per 400,000 g o f enzyme with p - C M B . U n d e r these c o n d i t i o n s a n d at p H 8.2 the Vrnax for fructose-6-P was not c h a n g e d while the Km was increased by a factor o f 5. O n the o t h e r hand, A T P titration curves showed no c h a n g e in Km b u t a significant decrease in Vmax. T h e effect o f such t i t r a t i o n on the kinetics at p H 6.9 was m o r e significant. These results are s u m m a r i z e d in T a b l e 4. T h e most interesting effect which can be seen was a decrease in c o o p e r a t i v i t y o f the enzyme as d e t e r m i n e d by the Hill coefficient. I n the absence o f A M P there is a decrease in the Hill coefficient from 3.4 to 2.2 as a result o f the sulfhydryl g r o u p titration. W h i l e in the presence o f the TABLE4 p]-] 6.7 BY p-CHLOROMERCUR1C BENZOATE(p-CMB) Enzyme was allowed to react with p-CMB (15 thiol groups per 400,000 g of enzyme). Activity was determined in the presence of I mM MgCI~ and 0.16 mM ATP. The concentration of AMP when present was 0.1 mM. Reaction was started by adding ATP. From the data of Froede and Mansour (27). M O D I F I C A T I O N OF K I N E T I C S OF P H O S P H O F R U U ' r O K I N A S E A T
gm Hill V max 5-AMP p-CMB (/~mole fructose-i,6-di-P/mg/min) (Fructose-6-P 10-*M) coefficient* -
--
+
--
--
+
+
--
55 48 19 32
3.8 0.8 4.2 1.0
3.4 2.0 2.2 1.0
*Hill coefficient was determined from the following equation: v log Vmax - v n logS ~ logK where S is substrate concentration, K is constant, v is observed activity, and Vrnaxis maximum activity extrapolated from a Lineweaver-Burk plot of the rates.
CONTROL OF PHOSPHOFRUCTOKINASE
4.5
adenylic nucleotide (or cyclic 3',5'-AMP) the Hill coefficient was decreased from 2.0 to 1.0. Thus positive effectors such as 5-AMP or cyclic 3',5'-AMP were capable of changing the cooperative kinetics ofp-CMB-treated enzyme to first order kinetics. This would indicate that the interaction between the fructose-6-P binding sites could be uncoupled when these thiol groups are titrated. In contrast, neither positive effector alone could completely convert the cooperative kinetics of the native enzyme to first order kinetics. The effects of sulfhydryl agents on the kinetics of phosphofructokinase described above do not appear to be due to the presence of these agents at the active site. This is supported by the fact that neither substrate protected these thiol groups against titration with sulfhydryl agents. Furthermore, when all the thiol groups were titrated with p-CMB, enzyme activity at pH 8.2 was only reduced to 50% of the original activity. The changes in the catalytic site, therefore, could be induced through conformational change in the protein. The role of sulfhydryl groups in determining the quaternary structure of phosphofructokinase is not unique. The cooperative binding of oxygen by hemoglobin is changed by modifying the sulfhydryl group at position 93 of the B-chain (29-31). In aspartate transcarbamylase enzyme activity is increased and it loses its cooperative properties when treated with divalent and monovalent mercurials (32). To|
t~
I
,
,
,
,
I
,
,
l\
°°Vg \ ,~
.~0~
o.o ~ o;
~ ~ 0 l ~
.;o
.;,
~o ~
i o
[ATP] (.raM)
~
f
F ~ .4,
.~o
FxG. 2 Plots of initial reaction velocity with respect to ATP concentration at pH 6.9 of native (O--O) and photo-oxidized enzyme (0--0). The insert shows a Lineweaver-Burkplot of the data. From Ahlfors and Mansour (12).
46
TAG E. MANSOUR
Desensitization of Sheep Heart Phosphofructokinase to Allosteric Control The discovery of the kinetics of phosphofructokinase introduced to the group of allosteric proteins a unique enzyme which has as an allosteric modifier one of its substrates. Both homotropic and heterotropic interactions are dependent on ATP concentrations. In an attempt to show conclusively that there are allosteric sites for ATP which are different from the catalytic sites, a modified form of the enzyme has recently been prepared by photo-oxidation in the presence of methylene blue (12). The photo-oxidized enzyme lost completely both its homotropic and heterotropic interaction. Neither ATP (Fig. 2)
-z
/
E
/
t
t .^ I "
, ~ 0 201~ I 0 .5
i
.1::,
>"1 _z.ol
l
K
..... i
Lo9 ~[~Cruc,o~-6 r P]x 104 ' I l' I I 10 1.5 2.0 2.5 3.0 ~,5 Irruc*ose -6 -P (m M) FiG. 3
Plots o f initial reaction velocity with respect to fructose-6-P at p H 6.9 of native ( O - - O ) and photo-oxidized ( O " - - Q ) enzyme. The insert shows plots derived from the Hill equation. F r o m Ahlfors and Mansour (12).
nor citrate could inhibit the enzyme. Furthermore, the Hill coefficient for fructose-6-P which was 3.15 for the native enzyme was reduced to 1.1 after photo-oxidation (Fig. 3). None of the activators (deinhibitors) had an effect on the modified enzyme. The Km for fructose-6-P at pH 6.9 was markedly reduced. All these effects were observed with only a slight reduction to the maximal enzyme activity. It therefore appears that photo-oxidation of the enzyme induces a specific conformation for the protein. This form of the enzyme shows no allosteric interaction, but the maximal activity is slightly affected. The question whether this form does not bind ATP at the allosteric sites is answered in the experiments described below.
47
CONTROL OF PHOSPHOFRUCTOKINASE
Binding Properties of Heart Phosphofructokinase Further evidence for the presence of allosteric sites which are different from the catalytic sites comes from recent studies on the binding properties of native and photo-oxidized enzyme (14). Binding studies were carried out using gel filtration technique (13). Some of these binding experiments are summarized in Table 5. While the native enzyme binds 14.4 moles of ATP per 400,000 g, TABLE 5 BINDING PROPERTIESOF NATIVE ENZYME AND OF ENZYME DESENSITIZEDTO ALLOSTERIC CONTROL BY PHOTO-OXIDATION Binding properties of phosphofructokinase were determined using the method of gel filtration described by Hummel and Dreyer (13). Data were calculated from doublereciprocal plots of substrate bound vs. free concentration of substrate. The dissociation constant (Ka) and the maximum theoretical number of binding sites per mole of protein (n) were determined by extrapolation of the reciprocal graphs. Lorenson and Mansour (14). Native Substrate ATP Citrate Fructose-6-P
Photo-oxidized
n
Ka
n
Ka
Mole/400,000 g 14.4 8.0 7.0
M 2 × l0-6 3 × l0 -r 2.1 × 10 -5
Mole/400,000 g 7.2 No binding 6.0
M 2.8 × 10-e No binding 2.0 × 10 -5
the photo-oxidized enzyme binds only 7.2 moles per 400,000 g. Citrate, which is not a substrate, but only an allosteric modifier binds to the native enzyme only to the extent of 8 moles per 400,000 g. On the other hand, the photooxidized enzyme does not bind any citrate. The binding of fructose-6-P was not affected by photo-oxidation. Both forms of the enzyme bind 7 moles of this substrate per 400,000 g. A conclusion can be drawn from this data that the native enzyme has 7 active sites and 7 allosteric sites. Both citrate and ATP bind at the same allosteric sites. Experiments failed to demonstrate a cooperative effect on the binding of fructose-6-P. This is possibly due to the fact that inorganic phosphate had to be used in these experiments in order to avoid non-specific binding. Under these conditions the kinetics for fructose-6-P are first order. Another possible reason for not demonstrating a cooperative effect is the fact that these studies were carried out in the absence of ATP to avoid starting enzyme reaction. The effect of pH on the binding of different ligands revealed some interesting findings which are summarized in Table 6. At a non-saturating ATP concentration of 10/zM the binding was markedly decreased at an alkaline pH. Furthermore, the enzyme did not bind citrate at an alkaline pH. The binding of fructose-6-P was not affected by pH. These experiments indicate that the binding to the allosteric sites is maximal at an acid pH. The results might explain the dependence ofallosteric interaction of the enzyme on an acidic pH.
48
TAG E. MANSOUR TABLE6 EFFECTOF pH ON THEBINDINGOF DIFFERENTSUBSTRATESBY PHOSPHOFRUCTOKINASE Binding of different substrates was determined at the indicated concentrations. From the data of Mansour and Lorenson (14). Substrate binding (molesper 400,000g) Substrate ATP 10/.zM ATP 10 t~M Citrate 5 /~M Citrate 5 ~M Fructose-6-P* Fructose-6-P*
pH
Native PFK
6 8 6 8 6.7 8.0
12.8 4.0 3.6 No binding 7.0 7.0
Photo-oxidized PFK 4
2 No binding No binding 6.4 6.4
*Maximal binding at infinite concentration of fructose-6-P was by extrapolation from reciprocal plots as described in Table 5.
In their studies on the binding properties of skeletal muscle phosphofructokinase Kemp and Krebs (33) reported that AMP, ADP and cyclic Y,5'-AMP bind at one common site per protomer (90,000 g). ATP can bind to the same site but it has a higher dissociation constant than that of the other nucleotides. The effect of the nucleotide activators, therefore, could be related to their competition with ATP for binding at this site. PHYSIOLOGICAL INTERPRETATIONS The above described experiments with both mammalian and fluke phosphofructokinases point to two main mechanisms which could be of importance from the standpoint of physiological regulation: I. Phosphofructokinase could be present in two forms, active and subactive. These two forms are interconvertible under certain conditions by an association-dissociation process. 2. Phosphofructokinase at pH 6.9 is allosterically inhibited by ATP, citrate and creatine phosphate. The inhibited enzyme can be activated by 5-AMP, cyclic 3',5'-AMP, A D P and P~. There is considerable evidence to support the conclusion that both mechanisms could be present in the liver fluke. The enzyme in the resting flukes is simply inactive, but is activated by serotonin. The activation is concomitant with an increase in glycolysis. The amine activates the enzyme and glycolysis in the cell-free extracts as well as in the intact organisms. The evidence here appears to be as strong as that available on the effect of epinephrine on mammalian liver phosphorylase. There is also considerable evidence that activation of the fluke enzyme is mediated through cyclic 3',5'-AMP although the details of the activating system have not yet been worked out because of
CONTROLOF PHOSPHOFRUCTOKINASE
49
the unavailability of a purified fluke enzyme. The fluke enzyme, once activated, is probably subject to allosteric control as described above. In the mammalian enzyme, although it can be reversibly converted to active and subactive forms, the evidence for the physiological importance of this process is incomplete. Freshly isolated tissues have always yielded a fully active enzyme. Our experience with these tissues is that they all show all the signs associated with anoxia. This simple fact throws some doubt on the assumption that enzyme levels of these tissues represent resting levels. The possibility, therefore, exists that in mammals, where the demand for oxidative metabolism is great, the enzyme is activated as soon as the tissues are removed from the animal. The role of phosphofructokinase in relation to anoxia has been postulated 0-3, 18). Evidence that the mammalian enzyme is regulated through its complex kinetic properties described above is indirect. An increase in glycolysis during anoxia is associated with a decrease in the intracellular levels of free glucose, glucose-6-P and fructose-6-P and a simultaneous increase in the concentration of fructose-l,6-di-P and the later glycolytic metabolites. These events coincide with an increase in the levels of 5'-AMP, ADP and Pt. An increase in the level of these agents could activate the enzyme by overcoming ATP inhibition of phosphofructokinase. Kinetic data suggest that phosphofructokinase can respond to what has been termed by Atkinson "'adenylate charge" (34, 35). Kinetic results reported by him with reactants and nucleotides at physiological concentrations showed that the enzyme could respond to quite small concentrations of AMP. A change in the adenylate charge from 1.0 to 0.9 results in an insignificant change in ATP concentration (negative modifier) but in a significant change in AMP levels (with a 4 mM adenylate pool the concentration of AMP is 0 at a charge of 1.0 and 100 tzM at a charge of 0.9). Citrate was shown to modulate this effect by increasing the steepness of the response curve to changes in the adenylate charge. Cyclic 3',5'-AMP acts directly on phosphofructokinase by releasing the enzyme from control through the adenylate system. This is compatible with our early observations on the fluke and the heart enzymes that the cyclic nucleotide renders the phosphofructokinase less susceptible to inhibition by ATP (5, I0). As it was pointed out by Atkinson (34) this hypothesis is subject to the limitation that we have no idea about the functional compartmentation in the living cell. Determinations of the levels of these substrates is always based on the assumption of their homogenous distribution within the cell. SUMMARY Kinetic and physical properties of phosphofructokinase from the liver fluke
Fasciola hepatica and from the mammalian heart were described. Evidence for the presence of an inactive-active conversion of the enzyme was presented.
50
TAG E. MANSOUR
T h e kinetic b e h a v i o r o f the enzyme was discussed in terms o f the M o n o d C h a n g e u x - W y m a n m o d e l for allosteric proteins. Studies were described elucidating the role o f thiol groups o n allosteric kinetics o f the enzyme. Evidence was presented showing that the allosteric site on the enzyme is different from the catalytic site. Binding properties o f the two sites to substrates a n d modifiers were presented. Possible physiological significance seen in all these properties was discussed in terms o f the regulation o f enzyme activity within the cell.
ACKNOWLEDGMENTS This research was s u p p o r t e d by U n i t e d States Public H e a l t h Service Research G r a n t A I O 4 2 1 4 from the N a t i o n a l Institute o f Allergy a n d Infectious Diseases; U n i t e d States Public H e a l t h Service C a r e e r D e v e l o p m e n t A w a r d G M 3848 f r o m the Division o f G e n e r a l M e d i c a l Sciences; a n d a G r a n t - i n - A i d from the A m e r i c a n H e a r t Association.
REFERENCES 1. Co F. CoRt, Phosphorylation of carbohydrates, pp. 175-189 in A Symposium on Respiratory Enzymes, University of Wisconsin Press, Madison (1942). 2. V. A. ENGEL'HARDTand N. E. SAKOV,The mechanism of the Pasteur effect, Biokhimiya 8, 9-36 (1943). 3. F. LYNEN, G. HARTMANN, K. F. NETTER and A. SCttUEGRAT, pp. 256-273 in Ciba Foundation Symposium on Regulation of Cell Metabolism (G. E. W. WOLSTENHOLME,ed.),
Little, Brown & Co., Boston (1959). 4. T. E. MANSOOR,Effect of serotonin on glycolysis in homogenates from the liver fluke, Fasciola hepatica, J. Pharmacol. Exptl. Therap. 135, 94-101 (1962). 5. T. E. MANSOURand J. M. MANSOUR, Effects of serotonin (5-hydroxytryptamine) and adenosine 3',5'-phosphate on phosphofructokinase from the liver fluke, Faseiola hepatica, J. Biol. Chem. 237, 629-634 (1962). 6. T. E. MANSOUR, E, W. SUTHERLAND,T. W. PALL and E. BUEDING, The effect of serotonin (5-hydroxytryptamine) on the formation of adenosine-Y,5'-phosphate by tissue particles from the liver fluke, Fasciola hepatica, J. Biol. Chem. 235, 466-470 (1960). 7. T. E. MANSOUR, N. WAKID and H. M. SPROUSE, Studies on heart phosphofructokinase: purification, crystallization and properties of sheep heart phosphofructokinase, J. BioL Chem. 241, 1512-1521 (1966). 8. D. B. STONEand T. E. MANSOUR,Phosphofruetokinase from the liver fluke, Fasciola hepatica. I. Activation by adenosine Y,5'-phosphate and by serotonin, Mol. Pharmacol.
3, 161-176 (1967). 9. D. B. STONEand T. E. MANSOUR,Phosphofructokinase from the liver fluke, Fasciola hepatica. II. Kinetic properties of the enzyme, Mol. Pharmacol. 3, 177-187 (1967). 10. T. E. MANSOUR,Studies on heart phosphofructokinase: purification, inhibition and activation, J. Biol. Chem. 238, 2285-2292 (1963). 11. T. E. MANSOUR, Phosphofructokinase II. Heart muscle, pp. 430-436 in Methods in Enzymology, Vol. IX (W. A. WOODS,ed.), Academic Press, Inc., New York 0968). 12. C. E. ArlLroRs and T. E. MANSOUR, Studies on heart phosphofructokinase: desensitization of the enzyme to adenosine triphosphate inhibition, J. Biol. Chem. 244, 1247-1251 (1969).
CONTROL OF PHOSPHOFRUCTOKINASE
51
13. J. P. HUMMELand W. J. D~vr.n, Measurement of proteln-binding phenomena by gel filtration, Biochim. Biophys. Acta 63, 530-532 (1962). 14. M. Y. LORENSONand T. E. MANSOLrP.,Studies on Heart phosphofructokinas¢: binding properties of native enzyme and of enzyme desensitized to allosteric control. J. Biol. Chem. 244, 6420-6431 (1969). 15. T. E. MANSOLrg,Studies on heart phosphofructokinas¢: active and inactive forms of the enzyme, J. Biol. Chem. 240, 2165-2172 (1965). 16. H. A. LARDYand R. E. PARKS, JR. Influence of ATP concentration on rates of some phosphorylation reactions, pp. 584-588 in Enzymes: Units of Biological Structure and Function (O. H. GAEnLER,ed.), Academic Press, Inc., New York (1956). 17. J. MONOD, J. WYMANand J.-P. CHANGEtrx, On the nature of allosteric transitions: a plausible model, J. Mol. Biol. 12, 88-118 (1965). 18. J. V. PASSOr,n~AU and O. H. LOWRY, Phosphofructokinase and the Pasteur effect, Biochem. Biophys. Res. Commun. 7, 10-15 (1962). 19. A. RAMAIAH,J. A. HATHAWAYand D. E. ATgrSSON, Adenylate as a metabolic regulator: effect on yeast phosphofructokinase kinetics, J. Biol. Chem. 239, 3619-3622 (1964). 20. A. PARM~t3GIA~ and R. H. BOWMAt~, Regulation of phosphofructokinas¢ activity by citrate in normal and diabetic muscle, Biochem. Biophys. Res. Commun. 12, 268-273 (1963). 21. P. B. GARLAND,P. J. RANDLEand E. A. NEWSHOLME,Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation, Nature 200, 169-170 (1963). 22. M. SALAS, E. Vn,rO~LA, M. SALASand A. Sot.s, Citrate inhibition of phosphofructokinase and the Pasteur effect, Biochem. Biophys. Res. Commun. 19, 371-376 (1965). 23. D. BLA~GY, H. Buc and J. MONOD, Kinetics of the allosteric interactions of phosphofructokinase from E. coli, J. Mol. Biol. 31, 13-35 (1968). 24. T. E. MANSOORand C. E. A~LFORS, Studies on heart phosphofructokinase: some kinetic and physical properties of the crystalline enzyme, J. Biol. Chem. 243, 2523-2533 (1968). 25. J. KRZANOWSKI and F. M. MA'I"SCI~NSKY, Regulation of phosphofructokinase by phosphocreatine and phosphorylated glycolytic intermediates, Biochem. Biophys. Res. Commun. 34, 816-823 (1969). 26. A. OTANI and T. E, MANSOUR,Unpublished observations. 27. H. C. FROF_DE,G. GERACÁand T. E. MANSOUR,Studies on heart phosphofructokinas~: thiol groups and their relationship to activity, d. Biol. Chem. 243, 6021-6029 (1968). 28. E. S. YOUNATHAN,V. PAETKAUand H. A. LARDY,Rabbit muscle phosphofructokinase reactivity and function of thiol groups, J. Biol. Chem. 243, 1603-1608 (1968). 29. A. R~G~S, The binding of N-ethylomaleimide by human hemoglobin and its effect upon the oxygen equilibrium, J. Biol. Chem. 236, 1948-1954 (1961). 30. R. BENESCHand R. E. BENESCH,The chemistry of the Bohr effect. I. The reaction of N-ethylmaleimide with the oxygen-linked acid groups of hemoglobin, J. Biol. Chem. 236, 405-410 (1961). 31. J. F. TAYLOn, E. Am~NI~n, M. Bgtmom and J. WYMAN,Studies on human hemoglobin treated with various sulfhydryl reagents, J. Biol. Chem. 241, 241-248 (1966). 32. J. C. G~a*.HARTand A. B. PARDEE,The enzymology of control by feedback inhibition, J. Biol. Chem. 237, 891-896 (1962). 33. R.. G. KEMP and E. G. Kilns, Binding of metabolites by phosphofructokinase, Biochemistry 6, 423-434 (1967). 34. D. E. ATKINSON, The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers, Biochemistry 7, 4030-4034 (1968). 35. L. C. SHEN,L. FALL,G. M. WALTONand D. E. ATKrNSON,Interaction between energy charge and metabolite modulation in the regulation of enzymes of amphibolic sequences. Phosphofructokinase and pyruvate dehydrogenas¢, Biochemistry 7, 4041--4045 (1968).