ARCHIVES
OF BIOCHEMISTRY
Specific
AND
BIOPHYSICS
178,
34-42 (1977)
Phosphorylation of Yeast Hexokinase Xylose and ATPMg Properties
of the Phosphorylated
LUIS CARLOS MENEZES’ Znstitut de Biochimie,
Universitt!
de Paris&d,
Induced
by
Form of the Enzyme
JULIO PUDLES
AND
Centre d’Orsay,
91405 Orsay, France
Received June 7, 1976 The inactivation of yeast hexokinase A (ATP:n-hexose-6-phosphotransferase, EC 2.7.1.D induced by n-xylose and ATPMg, is related to the phosphorylation of a serine residue. The phosphoenzyme can be specifically dephosphorylated and reactivated after treatment with alkaline phosphatase or when incubated with n-xylose or n-lyxose and ADPMg. The phosphorylation of hexokinase, as well as the dephosphorylation of the phosphoenzyme, are highly specific processes, depending on the formation of a quaternary complex (protein-pentose-nucleotide-Mg*+). Glucose or P-+!H,-ATP can inhibit the reactivation of the phosphoenzyme induced by xylose and ADPMg. The phosphorylation of the yeast hexokinase does not seem to affect the protein structure as seen by circular dichroism or fluorescence studies significantly. Binding studies, based on a quenching effect of ATP and glucose on the intrinsic fluorescence of the protein, show that both substrates can bind to the phosphohexokinase, but with slightly higher dissociation constants than with the native enzyme. Our results suggest that the essential serine residue is located in the active center region and that the phosphorylation of this residue represents a dead-end pathway in the course of the activation of the ATPase activity induced by xylose or lyxose.
DelaFuente et al. (1) have shown that Dxylose or n-lyxose, which are nonphosphorylatable inhibitors of glucose phosphorylation by yeast hexokinase, can enhance the ATPase activity of the enzyme. Furthermore DelaFuente (21observed that n-xylose has only a transitory activation effect, which is then followed by inactivation of the enzyme. These authors suggested that the binding of the two pentoses at the enzyme active site involves an “induced fit” which changes the protein conformation. In a preliminary communication, Cheng et al. (3) indicated that the inactivation induced by n-xylose was due to the phosphorylation of the protein. We have recently confirmed this result (4) and have shown that a serine residue per enzyme subunit is phosphorylated in the course of the inactivation process.
From the specificity and stoichiometry of the phosphorylation of the enzyme, it was thus important to determine whether the loss of hexokinase and ATPase activities were related to a change in the protein structure or to modifications of the substrate binding or catalytic sites. In this paper, we present results obtained from studies of the effects of different sugars and nucleotides on the reactivation of the phosphoenzyme, substrate-binding studies, and conformational studies on the modified protein. A preliminary report of part of this work has already been presented (5). EXPERIMENTAL
Baker’s yeast hexokinase A was purified as described by Rustum et aE. (6) with some modifications. The last two steps on DEAE-cellulose were replaced by a fractionation by isoelectric focusing, using a pH gradient of 0.5 pH unit (pH 4.75 to 5.25).
’ This and previously published work (4, 12, 15) have been carried out in partial fulflllement of a doctoral thesis submitted by L. C. Menezes. 34 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
PROCEDURE
Materials
PROPERTIES
OF PHOSPHORYLATED
Our enzyme preparation had a specific activity of 150 unitslmg at 25°C and was homogeneous both by disc electrophoresis and by ultracentrifugation. Pyruvate kinase and phosphoenolpyruvate dicyclohexylammonium salt were obtained from Boehringer Mannheim and alkaline phosphatase, from Worthington. The nucleotides were purchased from P-L Biochemicals. [+2PlATP was obtained from the Commissariat a l’Energie Atomique; P-y-CH,ATP, from Miles Laboratories; n-lyxose, from Fluka. n-Xylose and n-fructose were purchased from Merck; n-glucose and n-mannose, from Prolabo; glucose-6-phosphate was obtained from Calbiochemical; N-acetylglucosamine hydrochloride, from Sigma; 5,5’-dithiobis(2-nitrobenzoic acid) was obtained from Aldrich Chemicals Inc.; charcoal-impregnated filter paper, from Labo-Moderne (Paris); disc filter paper (2.4 cm diameter) 3MM from Whatman. All other chemicals were analytical reagent grade. Solutions were prepared in deionized distilled water.
Methods Enzymic assay. The hexokinase activity was measured by the potentiometric method, described by Hammes and Kochavi (7) on an automatic titrator radiometer Model 5 BRBL and ABU 11. The standard reaction mixture contained a final concentration of 5.7 mM ATP, 20 rnM MgC&, and 40 mM D-ghlCOSe; the volume of this solution used for each test was 1.5 ml. The enzyme utilized for this volume was between 0.8 and 15 pg of protein. The enzyme activity was tested at pH 8.5. The temperature of the system was maintained at 25°C. Protein concentration. Protein concentration was determined by absorption measurements at 280 nm. A value of 0.92 was taken for the absorbance of 1 mg/ ml of solution of hexokinase in a l-cm cuvette at 280 nm, as indicated by Lazarus et al. (8). Radioactivity measurements. The radioactivity of 32P was determined by measuring the Cerenkov radiation in 10 ml of distilled water (9), in a Packard liquid scintillation spectrometer. Polyethylene vials were used throughout. Inactivation of hexokinase with xylose in the presence of ATPMg. The incubation mixture was: 5 mM ATP, 15 mM MgCl,, 0.1 mM xylose, 50 mM phosphoenolpyruvate, and 10 pg of pyruvate kinase (1.5 units). The reaction was started by the addition of 0.1 to 1 mg of yeast hexokinase, and the final volume was 0.5 ml in 0.1 M tris(hydroxymethy1) methyl-2aminoethanesulfonic acid buffer at pH 7.0 and 25°C; aliquots were taken at different time intervals and assayed immediately for the enzyme activity. Estimation of the number of phosphate group incorporated. In order to determine the amount of phosphate incorporated during the inactivation process, the enzyme was incubated in the same con-
HEXOKINASE
35
ditions as described above (in the presence of xylose) and [-y-32P1ATP was added in the incubation mixture (0.13 mCi/ml). At the desired time intervals, 0.5-ml aliquots were taken, filtered through a Bio-Gel P-10 column (0.8 x 10 cm) previously equilibrated with 25 mM tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer solution at pH 7.0, and eluted with the same buffer. Fractions of 0.5 ml were collected and analyzed spectrophotometrically at 260 and 280 nm for nucleotides and protein, respectively, and for xylose by the method of Dubois et al. (10). Aliquots of each fraction were taken for radioactivity measurements. The calculations were based on a molecular weight of 51,000 for the enzyme subunit. The amount of radioactive phosphate incorporated was corrected from the dilution factor introduced by the ATP regeneration system, which contains nonlabeled phosphoenolpyruvate. In our experiment, the correction factor was never greater than 20% of the initial specific radioactivity. For the determination of the dilution factor, 5-~1 aliquots were taken from the inactivation mixture at different time intervals and diluted in 4 ml of 20 mM phosphate-buffered solution at pH 7.0. From this solution, 50 ~1 were spotted on filter paper discs (2.4 cm diameter) impregnated with active charcoal. The paper discs were then washed by gently stirring twice in 300 ml of phosphate-buffered solution, pH 7.0 (for 10 min each) to eliminate the radioactive inorganic phosphate, while the nucleotide was retained in the charcoal, after which they were immersed in 100 ml of 98% ethanol (5 min), in 100 ml of a mixture of ethanol-ethylether, 1:2 (5 min), and finally in 50 ml of ethylether (3 min). The paper discs were air-dried and the radioactivity was counted as described above. The semilogarithmic plot of the percentage decrease of [-@*PlATP radioactivity versus time of incubation in the inactivation system follows a linear process. Reactivation studies. The inactivated phosphoenzyme (0.8% residual activity) prepared as described above in the presence of n-xylose, ATPMg, phosphoenolpyruvate, and pyruvate kinase was filtered through a Bio-Gel P-10 column to eliminate the low molecular weight products, and the phosphoenzyme solution was used immediately for the reactivation studies. To measure reactivation, the phosphoenzyme was incubated in 25 mM tris(hydroxymethyl)methyl-2aminoethanesulfonic acid buffer, pH 7 at 25”C, in the presence of different sugars, substrates, or competitive inhibitors, at concentrations of 0.1 M, and ADPMg. ADP was present at a concentration of 21 mM, MgCl, at 25 mM, and the phosphoenzyme at a final concentration of 0.22 mgiml. At different time intervals, aliquots were taken and directly assayed for hexokinase activity. Dephosphorylation and reactivation of the [32Plphosphoenzyme. The 132Plphosphoenzyme ob-
36
MENEZES
tained after Bio-Gel P-10 filtration was incubated either with n-xylose and ADPMg or with Escherichia coli alkaline phosphatase. At different time intervals, two aliquots were taken to determine both the recovery of the hexokinase activity and the decrease in radioactivity of the 132Plphosphate-bound protein. For the determination of the decrease of 32P radioactivity on the phosphate-bound protein in the course of the reactivation process, the following procedure was used: 50 to 75 ~1 of the incubation mixture was spotted on filter paper discs (Whatman No. 3) 2.4 cm in diameter. The discs were immediately introduced into a beaker containing 500 ml of 6% trichloroacetic acid solution and kept in this solution and gently stirred for 10 min. After the trichloroacetic acid treatment, the filter paper discs were washed for 5 min in each of the following solutions: 200 ml of ethanol, 100 ml of ethanol-ethylether (1:2), and 100 ml of ethylether. The filter paper discs were then air-dried and the radioactivity counted. groups. Free sulihyEstimation of the sulfhydryl dry1 groups were titrated by the Ellmann spectrophotometric method in 0.1 M Tris buffer, pH 8 (11. The phosphoenzyme was obtained after incubation with xylose and ATPMg and filtration through a Bio-Gel P-10 column previously equilibrated with 0.1 M Tris buffer, pH 8. The protein concentration was 0.35 to 0.4 mg/ml. In all cases, a control experiment, without the addition of ATP, was carried out for comparison and is referred to as “native enzyme.” Fluorescence measurements. Fluorescence measurements were made on a Farrand MK-1 spectrofluorometer with a 150-W direct-current high-pressure xenon arc lamp as the light source and an RCA IP28 photomultiplier. For the studies of fluorescence spectra changes during enzyme inactivation, 250 pg of hexokinase was incubated in 2 ml of 0.1 M tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer, pH 7, containing 5 mM ATP, 15 mM MgC&, 0.1 M xylose, 50 mM phosphoenolpyruvate: and 1.5 units (10 pg) of pyruvate kinase. The reaction was started by the addition of ATP. At the desired time intervals, the fluorescence spectra were recorded (excitation at 295 nm) and aliquots were taken and immediatley assayed for hexokinase activity. The temperature was maintained at 22°C. A control experiment, without addition of ATP, was carried out. Binding studies. The binding of glucose and ATP to the enzyme was studied by fluorescence. The enzyme solution, with a protein concentration of 100 to 150 pg/ml in 50 mM Tris buffer, pH 7.5, containing 0.1 mM EDTA, was excited at 295 nm and the fluorescence emission was measured at 330 nm. The temperature was maintained at 25°C. The phosphorylated enzyme had less than 1% residual activity and was previously filtrated through a Bio-Gel P-10 column.
AND
PUDLES
For the ATP binding measurements, corrections were introduced based on the ATP quenching effect on a tryptophan solution having, at 280 nm, the same absorption as that of the protein solution used in the binding experiment. The ATP binding studies were performed in the presence of 5 mM glucose and in the absence of MgCl,. Circular dichroism. Circular dichroism measurements were performed with a Jouan II dichrograph. The enzyme (0.6 mg/ml) was incubated in 1 ml of 0.1 tris(hydroxymethyl)methyl-2-aminoethanesul&ic acid buffer, pH 7, containing 2.4 mM ATP, 6.8 mM MgCl,, 100 mM xylose, 45 mM phosphoenolpyruvate, and 1.5 units (10 pg) of pyruvate kinase. The mixture was introduced into a quartz cuvette, 0.203 mm in width, and the dichroic spectra were recorded at different time intervals. The temperature was maintained at 20°C. The enzyme activity was assayed at the end of the experiment. RESULTS
Inactivation of the Enzyme atpH 7.0 in the Presence of o-Xylose and ATPMg
In a previous publication (4), we showed that when hexokinase was incubated only in the presence of ATPMg and xylose, the rate of inactivation decreased progressively until a plateau was reached. If the incubation time was prolonged it was ob-
r-E
‘--I\.
TIME
(
hours)
FIG. 1. Inactivation of yeast hexokinase in the presence of xylose and [y3*PlATPMg, and incorporation of phosphate on the enzyme. Hexokinase was incubated in the presence of [Y-~~PIATP, MgCl,, xylose, and an ATP-regenerating system (phosphoenolpyruvate and pyruvate kinase) at pH 7.0 and 25°C. At different time intervals, aliquots were taken and the residual hexokinase activity was determined (0). The amount of szPi incorporated was determined after filtration through a Bio-Gel P-10 column (A). (For details see Methods.) The inset represents the relationship between the loss of hexokinase activity and the number of phosphate groups incorporated per enzyme subunit.
PROPERTIES 1
/-T
1s
3
45 TIME
OF PHOSPHORYLATED 100
----
6 796 (hours)
_.
9
,,
22
FIG. 2. Reactivation of the phosphohexokinase by incubation with xylose or lyxose and ADPMg. The hexokinase activity was measured at different time intervals during incubation of the phosphoenzyme in the presence of xylose (0) or lyxose (0) and ADPMg. The 32P release was followed when the 32Plabeled phosphoenzyme was incubated under the same conditions in the presence of xylose and ADPMg (0). (For experimental details, see Methods.)
served that hexokinase began to be reactivated, recovering virtually all of the original activity. However, if pyruvate kinase and phosphoenolpyruvate were added to the incubation mixture, providing a continuous regeneration of ATP, no plateau was reached after 26 h of reaction time and during this time interval 99.6% of the hexokinase and ATPase activities had been lost (Fig. 1). Under these conditions the inactivation kinetic followed a first-order process until 90% of the enzyme activity (h = 9 x 10e3min-‘) was lost. Moreover, the native enzyme treated under the same conditions but in absence of ATP, lost only 5% of the initial activity. The incorporation of 32Pinto the protein was parallel to the loss of the enzyme activity. Moreover, the complete inactivation of the enzyme was obtained when one phosphate group was incorporated per enzyme subunit (molecular weight, 51,000) (insert Fig. 1). Reactivation
of the Modified
Enzyme
The inactivated enzyme (less than 1% activity) can be reactivated after gel filtration by incubation with ADPMg and Dxylose or n-lyxose (Fig. 2). The reactivation is in both cases a first-order process, nearly three times faster in the presence of xylose than in the presence of lyxose (h = 8.3 x 10e3 and 3 x 10m3min’, respectively). 32Pwas released from the protein
37
HEXOKINASE
in the presence of xylose at the same rate as the enzyme was reactivated (Fig. 2). The reactivation effect is quite specific. As shown in Table I, both the nucleotide (ADP) and the metal were essential requirements for the reactivation, as was the presence of one of the sugars cited above, xylose or lyxose. All other sugars assayed, whether they were substrates or products in the hexokinase reaction, were unable to induce the reactivation process. Moreover, the enzyme was not reactivated if AMP was substituted for ADP (Table I). On the other hand, when glucose was added to the reactivating system containing xylose and ADPMg, we observed an inhibitory effect on the enzyme reactivation rate (Fig. 3). Moreover, fly-CH,-ATP, an analog of ATP, has a similar inhibitory effect on the enzyme reactivation. The addition of P-y-CH,-ATP to the reactivating system at a concentration of 0.1 M with an equivalent addition of MgCl, reduces the rate of enzyme reactivation to 50% (results not shown). The 132Plphosphoenzyme also can be reactivated by incubation with alkaline TABLE
I
REACTIVATION OF THE PHOSPHOHEXOKINASE PRESENCE OF DIFFERENT LIGAND@
Additions
None Xylose Xylose and ADP Xylose, MgCl,, and ADP MgCl, and ADP Xylose, MgCl,, and AMP Lyxose Lyxose, MgCl,, and ADP Glucose Glucose, MgCl,, and ADP Glucosamme, MgCl,, and ADP Mannose, MgCl,, and ADP Fructose, MgCl,, and ADP Glucose-6-P, MgCl,, and ADP
IN THE
Recovered activity woo) 4.1 1.8 3.0 100.0 2.2 1.1 1.2 94.0 1.0 1.2 1.3 1.3 1.0 1.0
a After filtration through a Bio-Gel P-10 column the phosphoenzyme having 1% residual activity was incubated at 25°C in 25 mM tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer, pH 7, in the presence or absence of sugars (0.1 M), nucleotides (21 mM), and MgCl, (25 mM). The enzyme activity was assayed after 23 h of incubation. (For details, see Methods.)
38
MENEZES AND PUDLES
TIME
( min
)
FIG. 3. Inhibitory effect of glucose on the rate of phosphoenxyme reactivation. The phosphohexokinase was incubated at 25°C in the presence of 0.1 M xylose, 43 mM ADP, and 50 mM MgCl,. Glucose was added to a final concentration of 0.3 (0), 0.6 (A), 0.9 (O), 1.2 (O), or 1.5 (A) mM. A control was done without addition of glucose (W. In this figure, the straight lines represent the kinetics of the enzyme reactivation. A”, relative activity of the native enzyme; Al, relative activity of the phosphoenzyme at different time intervals.
phosphatase. Under these conditions, the [32P]phosphoenzyme was rapidly reactivated with a decrease in 32Pradioactivity of the phosphate-bound protein (Fig. 4). CONFORMATIONAL
STUDIES
Reactivity of the Sulfiydryl Groups of the Native and Phosphorylated Hexokinase Hexokinase A has four sulthydryl groups per subunit, one fast- and three slow-reacting groups, when titrated with Nbs, (12).2 Similar results were obtained for the phosphorylated enzyme, having 12% residual activity, after 6 h of incubation with xylose and ATPMg and elimination of the nucleotide by gel filtration through a Bio-Gel P-10 column previously equilibrated with 0.1 M xylose solution in 0.1 M Tris buffer, pH 8. As shown in Fig. 5, for both the native enzyme and the modified enzyme, the reaction of the sulfhydryl groups with Nbs, is biphasic, but the reaction rate of the three slow-reacting sulfhydry1 groups in the phosphoenzyme (k = 0.076 min-‘1 was enhanced by a factor of 3.5 as compared with the rate for the native enzyme (k = 0.022 min-‘). 2 Abbreviations used: Nbs,, 5,5’-dithiobis(2-nitrobenzoic acid); DEAE, diethylaminoethyl.
If the incubation with xylose and ATPMg was prolonged until the loss of 98% of the activity and the reactivity of the sulfhydryl groups were measured after elimination of the nucleotide and the sugar by gel filtration, the reactivity of the three “slow-reacting” sulfhydryl groups was found to be enhanced 10 times in the modified enzyme as compared to the native enzyme (see Table II). However, the addition of xylose or glucose significantly reduces the reactivity of the slow-reacting sulfhydryl groups (13) both in the native
‘Y’“”
TIME
(min)
FIG. 4. Reactivation of the phosphohexokinase by treatment with alkaline phosphatase. This figure represents both the recovery of the enzyme activity (W) and the release of 32P (0) from the 32P-labeled phosphoenzyme (0.25 mglml) incubated with alkaline phosphatase (1 mglml) at 3o”C, in 0.1 M Tris buffer, pH 7.6, containing 0.4 M NaCl.
30
60
90
120
160
180
TIME (min) FIG. 5. Determination of sulthydryl groups of the native and phosphoenzyme using Nbs, in the presence of xylose. Native enzyme (A); phosphoenzyme with 12% residual activity (0) (for experimental details, see Methods). The inset represents the semilogarithmic plot of the residual sulfhydryl groups versus time in the reaction with Nbs,.
PROPERTIES TABLE
OF PHOSPHORYLATED
II
RATE CONSTANTS OF THE “SLOW REACTING” SULFHYDRYL GROUP WITH Nbs, IN THE NATIVE HEXOKINASE AND IN THE PHOSPHOENZYMIF Rate constant (min-9 Additions
None D-Xylose (100 rnM) D-Glucose (5 m&f)
Native enzyme
Phosphoenzymeb
0.033 0.024 0.003
0.342 0.228 0.014
a Proteins were in 0.1 M Tris buffer, pH 8.0 and the reaction was carried out at the same pH. b Phosphoenzyme obtained after gel filtration and having 2% residual activity. (For details, see Methods.)
HEXOKINASE
39
sion spectra for the phosphorylated enzyme (not shown). From the chemical reactivity of the sulfhydryl groups of the phosphorylated hexokinase, there was an indication of a conformational change on the protein as compared with the native enzyme. However, using the fluorescence technique, we were unable to detect a significant perturbation of the protein structure due to the phosphorylation of the enzyme. It was thus important to determine whether a conformational change could be observed during the inactivation process by circular dichroism in the far ultraviolet region. By this method we could not observe any spectral difference between the native and phosphorylated enzyme, which showed a 94% loss of initial enzyme activity.
and in the phosphorylated enzyme. Indeed, as can be seen in Table II, the rate constant for the reaction of the sulfhydryl groups was reduced by a factor of 1.4 or 11 Binding of Glucose and ATP to the Native by the addition of xylose or glucose, respecand Phosphorylated Enzyme tively, to the native enzyme, and by a Zewe et al. (16) have shown that the factor of 1.5 or 24 if the same ligands were hexokinase fluorescence is quenched by added to the phosphorylated enzyme. the presence of glucose, ATP, and ATPMg. The dissociation constants that they obIntrinsic Fluorescence and Circular Ditained were similar to those obtained kichroism Studies of the Native and Phosnetically. Their results indicated that the phorylated Hexokinase fluorescence quenching was the result of The change in the reactivity of the binding of the substrates at the enzyme active site. sulfhydryl groups due to the inactivation process suggested that the conformation of the protein was affected by the phosphorylation of the essential serine residue. Since fluorescence methods have been found to be particularly sensitive to molecular changes in conformation of proteins (14, 15), it was interesting to observe whether, using this method, a conformational transformation of the protein in the course of the inactivation process could be detected. The kinetics of the inactivation process TIME ,mm, was followed by measurement of the loss of FIG. 6. Intrinsic fluorescence of hexokinase and the enzyme activity and by the change on the fluorescence emission at 330 nm (ex- loss in enzyme activity during incubation with xycited at 295 nm). As can be seen in Fig. 6, lose and ATPMg. The relative fluorescence intensity at 330 nm was measured during the incubation of only a slight decrease of fluorescence was hexokinase with xylose (0) or xylose and ATPMg detected during the reaction course as (0). Throughout the experiment, aliquots were compared with results for the native en- taken and assayed immediately for hexokinase aczyme under similar conditions but without tivity (A). F,,, fluorescence intensity at the time ATP addition. Moreover, we did not ob- zero; F, fluorescence intensity at different incubaserve any change in the maximum emis- tion times. (For experimental details, see Methods.)
40
MENEZES
AND
PUDLES
ciation constants for ATP with the binary complex were 0.55 and 1.74 mM, respectively, for the native and modified enzyme. Our dissociation constant values obtained with the native enzyme are somewhat higher than those observed by Zewe et al. (16). However, from our results it is evident that the substrates were capable of binding to the phosphoenzyme, though with a higher Kd. -4
-2
2
4
6
l/
8
10
I GLUCOSE]
12
I4
(mM-‘l
FIG. 7. Binding of glucose and ATP to the native and phosphoenzyme studied by fluorescence. (A) A double-reciprocal plot of the quenching effect of glucose on the enzyme fluorescence at 330 nm (excited at 295 nm) versus the glucose concentration. Native enzyme (A); phosphoenzyme (0) (see Methods). (B) A double-reciprocal plot of the quenching of ATP on the enzyme fluorescence at 330 nm (excited at 295 nm) versus ATP concentration. The enzyme was in the presence of 5 mM glucose. Native enzyme (A); phosphoenzyme (0).
We observe that the quenching effect which results from the binding of glucose is independent of the quenching effect induced by ATP. We were able to determine by the fluorescence method the binding constant for both glucose and ATP on the binary complex (enzyme-glucose). Figures 7A and B show double-reciprocal plots of the variation in the relative fluorescence intensity as a function of the ligand concentration. The dissociation constant were calculated from a linear regression analysis and the values found for glucose were 0.44 and 0.63 mM, respectively, whereas the disso-
DISCUSSION
In a preceding paper (4) we showed that the inactivation of yeast hexokinase induced by n-xylose and ATPMg is directly related to the phosphorylation of one serine residue per enzyme subunit. The phosphoenzyme, when incubated in the presence of xylose and ADPMg, can be dephosphorylated and reactivated with the same rate constant as that found for the inactivation process. Moreover, the same reactivation effect is also observed when the phosphoenzyme is incubated in the presence of lyxose and ADPMg. Except for the effect of alkaline phosphatase, which hydrolyzes the protein-bound phosphate and reactivates the phosphoenzyme, the inactivation and reactivtion processes are strictly dependent on the formation of a quaternary complex between the protein, xylose or lyxose, and ATPMg for the inactivation or ADPMg for the reactivation process. Sols et al. (17) as well as Rudolph and Fromm (18) have shown that lyxose and xylose are competitive inhibitors of the hexokinase activity. On the other hand, Anderson and Steitz (19) concluded from their low-resolution X-ray diffraction studies yeast hexokinase-substrate complexes that there is only one sugar binding site per enzyme subunit and that xylose in the presence of ADPMg binds to the same site as glucose. Moreover, DelaFuente observed that glucose inhibits the inactivation process by xylose and ATPMg, whereas in this paper we show that glucose or p-y-CH,-ATP inhibit the reactivation process. All of these data suggest that phosphorylation occurs at the level of the enzyme-active center region. Neverthe-
PROPERTIES
OF PHOSPHORYLATED
less, the nonreactivation of the phosphoenzyme by the sugar substrates in the absence or presence of ADPMg indicates that this modified form of the enzyme is not the controversial “phosphoryl-enzyme” intermediate of the hexokinase reaction (20). From the reactivation studies and fluorescence binding studies, it is quite clear that the substrate-binding sites on the enzyme do not seem to be significantly affected by the phosphorylation process. Even though the I-Cdvalues for glucose and ATP have been increased 1.2- and 3-fold, respectively, this increase could not explain the complete loss of the enzyme activity, since the enzyme assays were done with a substrate concentration much higher than the I& values, On the other hand, the conformational studies which were done by circular dichroism and fluorescence did not indicate any significant perturbation of the protein structure in the course of the phosphorylation. Jones et al. (21) and Otieno et al. (22), from their studies on the reactivity of the sulfhydryl groups of hexokinase B, suggested the possibility that some of the slow-reacting sulfhydryl groups could be in the proximity of the enzyme active center region. Then the observed change in the reactivity to Nbs, of the “slow reacting” sulfhydryl groups of the phosphoenzyme as compared with the native enzyme might quite possibly reflect a small perturbation in the vicinity of the enzyme-active center. However, this limited perturbation in the protein structure does not affect the formation of the quaternary complex of the phosphoenzyme with xylose or lyxose and ADPMg in the reactivation process, suggesting that the geometry of the enzymeactive site is preserved in the phosphohexokinase . The above results indicate that the reactive serine residue does not seem to be directly involved in the substrate binding sites or in the maintenance of the protein structure. Moreover, these results suggest that the phosphorylation of yeast hexokinase probably results from a specific conformational change of the enzyme active site induced by xylose or lyxose and
HEXOKINASE
41
ATPMg. This conformational change might modify the microenvironment of the serine residue, decreasing its PK, and consequently increasing its nucleophilicity, and furthermore, orienting the primary hydroxyl group into the vicinity of the yphosphate group of ATP. On the other hand, from the data described above we cannot exclude the possibility that this serine residue is implicated in the mechanism of action of yeast hexokinase. For the moment, we have no idea of the eventual physiological significance (if any) of this inactivating phosphorylation process; nonetheless, it represents an interesting and specific method for labeling an essential residue of hexokinase. ACKNOWLEDGMENTS This work was supported by grants from the Centre National de la Recherche Scientifique (Equipe de Recherche No. 142), Delegation Generale a la Recherche Scientitique et Technique (Contract No. 757.0188). We are grateful to Mrs. F. E. Tejtel and Dr. J. van Heijenoort for revising this manuscript. REFERENCES 1. DELAF’UENTE, G., LAGUNAS, R., AND SOLS, A. (1970) Eur. J. Biochem. 16, 226-233. 2. DELAFUENTE, G. (1970) Eur. J. Biochem. 16, 240-243. 3. CHENG, L. Y., INAGAMI, T., AND COLOWICK, S. P. (1973) Fed. Proc. 667, Abstract. 4. MENEZES, L. C., AND PUDLES, J. (1976) Eur. J. Biochem. 65, 41-47. 5. MENEZES, L. C. (1975) FEBS Meeting, Paris, Abstract 678. 6. RUSTUM, Y. M., RAMEL, A. H., AND BARNARD, E. A. (1971) Prep. Biochem. 1, 309-329. 7. HAMMES, G. G., AND KOCHAVI, D. (1962) J. Amer. Chem. Sot. 84, 2069-2073. 8. LAZARUS, N. R., RAMEL, A. H., RUSTUM, Y. R., AND BARNARD, E. A. (1966) Biochemistry 5, 4003-4016. 9. CLAUSEN, T. (1968)Anal. Biochem. 22,70-73. 10. DUBOIS, M., GILLES, K., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956)Anal. Chem. 28, 350-356. 11. ELLMANN, G. L. (1959) Arch. Biochem. 82, 7077. 12. ROSSI, A., MENEZES, L. C., AND PUDLES, J. (1975) Eur. J. Biochem. 59, 423-432. 13. GROUSELLE, M., (1969) Thesis “Doctorat de 3eme
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MENEZES
Cycle,” Universitk de Paris, Centre d’Orsay. 14. ANDERSON, S., AND WEBER, G. (1966)Arch. Biothem. Biophys. 116, 207-223. 15. MENEZES, L. C., GROUSELLE, M., ANDPUDLES, J. (1972) Eur. J. Biochem. 30, 81-92. 16. ZEWE, V., FROMM, H. J., AND FABIANO, R. (1964) J. Biol. Chem. 239, 1625-1634. 17. Sons, A., DELAFUENTE, G., VILLAR-PALLASI, C., AND ASENSIO, G. (1958) Biochim. Biophys. Acta 30, 92-101. 18. RUDOLPH, F. B., AND FROMM, H. J. (1971) J.
AND
PUDLES
Biol. Chem. 246, 2104-2110. 19. ANDERSON, W. F., AND STEITZ, T. A. (1975) J. Mol. Biol. 92, 279-287. 20. WALSH, C. T., JR., AND SPECTOR, L. B. (1971) Arch. Biochem. Biophys. 145, l-5. 21. JONES, J. G., OTIENO, S., BARNARD, E. A., AND BHARGAVA, A. K. (1975) Biochemistry 14, 2396-2402. 22. OTIENO, S., BHARGAVA, A. K., BARNARD, E. A., AND RAMEL, A. H. (1975) Biochemistry 14, 2403-2410.
OF BIOCHEMISTRY
Specific
AND
BIOPHYSICS
178,
34-42 (1977)
Phosphorylation of Yeast Hexokinase Xylose and ATPMg Properties
of the Phosphorylated
LUIS CARLOS MENEZES’ Znstitut de Biochimie,
Universitt!
de Paris&d,
Induced
by
Form of the Enzyme
JULIO PUDLES
AND
Centre d’Orsay,
91405 Orsay, France
Received June 7, 1976 The inactivation of yeast hexokinase A (ATP:n-hexose-6-phosphotransferase, EC 2.7.1.D induced by n-xylose and ATPMg, is related to the phosphorylation of a serine residue. The phosphoenzyme can be specifically dephosphorylated and reactivated after treatment with alkaline phosphatase or when incubated with n-xylose or n-lyxose and ADPMg. The phosphorylation of hexokinase, as well as the dephosphorylation of the phosphoenzyme, are highly specific processes, depending on the formation of a quaternary complex (protein-pentose-nucleotide-Mg*+). Glucose or P-+!H,-ATP can inhibit the reactivation of the phosphoenzyme induced by xylose and ADPMg. The phosphorylation of the yeast hexokinase does not seem to affect the protein structure as seen by circular dichroism or fluorescence studies significantly. Binding studies, based on a quenching effect of ATP and glucose on the intrinsic fluorescence of the protein, show that both substrates can bind to the phosphohexokinase, but with slightly higher dissociation constants than with the native enzyme. Our results suggest that the essential serine residue is located in the active center region and that the phosphorylation of this residue represents a dead-end pathway in the course of the activation of the ATPase activity induced by xylose or lyxose.
DelaFuente et al. (1) have shown that Dxylose or n-lyxose, which are nonphosphorylatable inhibitors of glucose phosphorylation by yeast hexokinase, can enhance the ATPase activity of the enzyme. Furthermore DelaFuente (21observed that n-xylose has only a transitory activation effect, which is then followed by inactivation of the enzyme. These authors suggested that the binding of the two pentoses at the enzyme active site involves an “induced fit” which changes the protein conformation. In a preliminary communication, Cheng et al. (3) indicated that the inactivation induced by n-xylose was due to the phosphorylation of the protein. We have recently confirmed this result (4) and have shown that a serine residue per enzyme subunit is phosphorylated in the course of the inactivation process.
From the specificity and stoichiometry of the phosphorylation of the enzyme, it was thus important to determine whether the loss of hexokinase and ATPase activities were related to a change in the protein structure or to modifications of the substrate binding or catalytic sites. In this paper, we present results obtained from studies of the effects of different sugars and nucleotides on the reactivation of the phosphoenzyme, substrate-binding studies, and conformational studies on the modified protein. A preliminary report of part of this work has already been presented (5). EXPERIMENTAL
Baker’s yeast hexokinase A was purified as described by Rustum et aE. (6) with some modifications. The last two steps on DEAE-cellulose were replaced by a fractionation by isoelectric focusing, using a pH gradient of 0.5 pH unit (pH 4.75 to 5.25).
’ This and previously published work (4, 12, 15) have been carried out in partial fulflllement of a doctoral thesis submitted by L. C. Menezes. 34 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
PROCEDURE
Materials
PROPERTIES
OF PHOSPHORYLATED
Our enzyme preparation had a specific activity of 150 unitslmg at 25°C and was homogeneous both by disc electrophoresis and by ultracentrifugation. Pyruvate kinase and phosphoenolpyruvate dicyclohexylammonium salt were obtained from Boehringer Mannheim and alkaline phosphatase, from Worthington. The nucleotides were purchased from P-L Biochemicals. [+2PlATP was obtained from the Commissariat a l’Energie Atomique; P-y-CH,ATP, from Miles Laboratories; n-lyxose, from Fluka. n-Xylose and n-fructose were purchased from Merck; n-glucose and n-mannose, from Prolabo; glucose-6-phosphate was obtained from Calbiochemical; N-acetylglucosamine hydrochloride, from Sigma; 5,5’-dithiobis(2-nitrobenzoic acid) was obtained from Aldrich Chemicals Inc.; charcoal-impregnated filter paper, from Labo-Moderne (Paris); disc filter paper (2.4 cm diameter) 3MM from Whatman. All other chemicals were analytical reagent grade. Solutions were prepared in deionized distilled water.
Methods Enzymic assay. The hexokinase activity was measured by the potentiometric method, described by Hammes and Kochavi (7) on an automatic titrator radiometer Model 5 BRBL and ABU 11. The standard reaction mixture contained a final concentration of 5.7 mM ATP, 20 rnM MgC&, and 40 mM D-ghlCOSe; the volume of this solution used for each test was 1.5 ml. The enzyme utilized for this volume was between 0.8 and 15 pg of protein. The enzyme activity was tested at pH 8.5. The temperature of the system was maintained at 25°C. Protein concentration. Protein concentration was determined by absorption measurements at 280 nm. A value of 0.92 was taken for the absorbance of 1 mg/ ml of solution of hexokinase in a l-cm cuvette at 280 nm, as indicated by Lazarus et al. (8). Radioactivity measurements. The radioactivity of 32P was determined by measuring the Cerenkov radiation in 10 ml of distilled water (9), in a Packard liquid scintillation spectrometer. Polyethylene vials were used throughout. Inactivation of hexokinase with xylose in the presence of ATPMg. The incubation mixture was: 5 mM ATP, 15 mM MgCl,, 0.1 mM xylose, 50 mM phosphoenolpyruvate, and 10 pg of pyruvate kinase (1.5 units). The reaction was started by the addition of 0.1 to 1 mg of yeast hexokinase, and the final volume was 0.5 ml in 0.1 M tris(hydroxymethy1) methyl-2aminoethanesulfonic acid buffer at pH 7.0 and 25°C; aliquots were taken at different time intervals and assayed immediately for the enzyme activity. Estimation of the number of phosphate group incorporated. In order to determine the amount of phosphate incorporated during the inactivation process, the enzyme was incubated in the same con-
HEXOKINASE
35
ditions as described above (in the presence of xylose) and [-y-32P1ATP was added in the incubation mixture (0.13 mCi/ml). At the desired time intervals, 0.5-ml aliquots were taken, filtered through a Bio-Gel P-10 column (0.8 x 10 cm) previously equilibrated with 25 mM tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer solution at pH 7.0, and eluted with the same buffer. Fractions of 0.5 ml were collected and analyzed spectrophotometrically at 260 and 280 nm for nucleotides and protein, respectively, and for xylose by the method of Dubois et al. (10). Aliquots of each fraction were taken for radioactivity measurements. The calculations were based on a molecular weight of 51,000 for the enzyme subunit. The amount of radioactive phosphate incorporated was corrected from the dilution factor introduced by the ATP regeneration system, which contains nonlabeled phosphoenolpyruvate. In our experiment, the correction factor was never greater than 20% of the initial specific radioactivity. For the determination of the dilution factor, 5-~1 aliquots were taken from the inactivation mixture at different time intervals and diluted in 4 ml of 20 mM phosphate-buffered solution at pH 7.0. From this solution, 50 ~1 were spotted on filter paper discs (2.4 cm diameter) impregnated with active charcoal. The paper discs were then washed by gently stirring twice in 300 ml of phosphate-buffered solution, pH 7.0 (for 10 min each) to eliminate the radioactive inorganic phosphate, while the nucleotide was retained in the charcoal, after which they were immersed in 100 ml of 98% ethanol (5 min), in 100 ml of a mixture of ethanol-ethylether, 1:2 (5 min), and finally in 50 ml of ethylether (3 min). The paper discs were air-dried and the radioactivity was counted as described above. The semilogarithmic plot of the percentage decrease of [-@*PlATP radioactivity versus time of incubation in the inactivation system follows a linear process. Reactivation studies. The inactivated phosphoenzyme (0.8% residual activity) prepared as described above in the presence of n-xylose, ATPMg, phosphoenolpyruvate, and pyruvate kinase was filtered through a Bio-Gel P-10 column to eliminate the low molecular weight products, and the phosphoenzyme solution was used immediately for the reactivation studies. To measure reactivation, the phosphoenzyme was incubated in 25 mM tris(hydroxymethyl)methyl-2aminoethanesulfonic acid buffer, pH 7 at 25”C, in the presence of different sugars, substrates, or competitive inhibitors, at concentrations of 0.1 M, and ADPMg. ADP was present at a concentration of 21 mM, MgCl, at 25 mM, and the phosphoenzyme at a final concentration of 0.22 mgiml. At different time intervals, aliquots were taken and directly assayed for hexokinase activity. Dephosphorylation and reactivation of the [32Plphosphoenzyme. The 132Plphosphoenzyme ob-
36
MENEZES
tained after Bio-Gel P-10 filtration was incubated either with n-xylose and ADPMg or with Escherichia coli alkaline phosphatase. At different time intervals, two aliquots were taken to determine both the recovery of the hexokinase activity and the decrease in radioactivity of the 132Plphosphate-bound protein. For the determination of the decrease of 32P radioactivity on the phosphate-bound protein in the course of the reactivation process, the following procedure was used: 50 to 75 ~1 of the incubation mixture was spotted on filter paper discs (Whatman No. 3) 2.4 cm in diameter. The discs were immediately introduced into a beaker containing 500 ml of 6% trichloroacetic acid solution and kept in this solution and gently stirred for 10 min. After the trichloroacetic acid treatment, the filter paper discs were washed for 5 min in each of the following solutions: 200 ml of ethanol, 100 ml of ethanol-ethylether (1:2), and 100 ml of ethylether. The filter paper discs were then air-dried and the radioactivity counted. groups. Free sulihyEstimation of the sulfhydryl dry1 groups were titrated by the Ellmann spectrophotometric method in 0.1 M Tris buffer, pH 8 (11. The phosphoenzyme was obtained after incubation with xylose and ATPMg and filtration through a Bio-Gel P-10 column previously equilibrated with 0.1 M Tris buffer, pH 8. The protein concentration was 0.35 to 0.4 mg/ml. In all cases, a control experiment, without the addition of ATP, was carried out for comparison and is referred to as “native enzyme.” Fluorescence measurements. Fluorescence measurements were made on a Farrand MK-1 spectrofluorometer with a 150-W direct-current high-pressure xenon arc lamp as the light source and an RCA IP28 photomultiplier. For the studies of fluorescence spectra changes during enzyme inactivation, 250 pg of hexokinase was incubated in 2 ml of 0.1 M tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer, pH 7, containing 5 mM ATP, 15 mM MgC&, 0.1 M xylose, 50 mM phosphoenolpyruvate: and 1.5 units (10 pg) of pyruvate kinase. The reaction was started by the addition of ATP. At the desired time intervals, the fluorescence spectra were recorded (excitation at 295 nm) and aliquots were taken and immediatley assayed for hexokinase activity. The temperature was maintained at 22°C. A control experiment, without addition of ATP, was carried out. Binding studies. The binding of glucose and ATP to the enzyme was studied by fluorescence. The enzyme solution, with a protein concentration of 100 to 150 pg/ml in 50 mM Tris buffer, pH 7.5, containing 0.1 mM EDTA, was excited at 295 nm and the fluorescence emission was measured at 330 nm. The temperature was maintained at 25°C. The phosphorylated enzyme had less than 1% residual activity and was previously filtrated through a Bio-Gel P-10 column.
AND
PUDLES
For the ATP binding measurements, corrections were introduced based on the ATP quenching effect on a tryptophan solution having, at 280 nm, the same absorption as that of the protein solution used in the binding experiment. The ATP binding studies were performed in the presence of 5 mM glucose and in the absence of MgCl,. Circular dichroism. Circular dichroism measurements were performed with a Jouan II dichrograph. The enzyme (0.6 mg/ml) was incubated in 1 ml of 0.1 tris(hydroxymethyl)methyl-2-aminoethanesul&ic acid buffer, pH 7, containing 2.4 mM ATP, 6.8 mM MgCl,, 100 mM xylose, 45 mM phosphoenolpyruvate, and 1.5 units (10 pg) of pyruvate kinase. The mixture was introduced into a quartz cuvette, 0.203 mm in width, and the dichroic spectra were recorded at different time intervals. The temperature was maintained at 20°C. The enzyme activity was assayed at the end of the experiment. RESULTS
Inactivation of the Enzyme atpH 7.0 in the Presence of o-Xylose and ATPMg
In a previous publication (4), we showed that when hexokinase was incubated only in the presence of ATPMg and xylose, the rate of inactivation decreased progressively until a plateau was reached. If the incubation time was prolonged it was ob-
r-E
‘--I\.
TIME
(
hours)
FIG. 1. Inactivation of yeast hexokinase in the presence of xylose and [y3*PlATPMg, and incorporation of phosphate on the enzyme. Hexokinase was incubated in the presence of [Y-~~PIATP, MgCl,, xylose, and an ATP-regenerating system (phosphoenolpyruvate and pyruvate kinase) at pH 7.0 and 25°C. At different time intervals, aliquots were taken and the residual hexokinase activity was determined (0). The amount of szPi incorporated was determined after filtration through a Bio-Gel P-10 column (A). (For details see Methods.) The inset represents the relationship between the loss of hexokinase activity and the number of phosphate groups incorporated per enzyme subunit.
PROPERTIES 1
/-T
1s
3
45 TIME
OF PHOSPHORYLATED 100
----
6 796 (hours)
_.
9
,,
22
FIG. 2. Reactivation of the phosphohexokinase by incubation with xylose or lyxose and ADPMg. The hexokinase activity was measured at different time intervals during incubation of the phosphoenzyme in the presence of xylose (0) or lyxose (0) and ADPMg. The 32P release was followed when the 32Plabeled phosphoenzyme was incubated under the same conditions in the presence of xylose and ADPMg (0). (For experimental details, see Methods.)
served that hexokinase began to be reactivated, recovering virtually all of the original activity. However, if pyruvate kinase and phosphoenolpyruvate were added to the incubation mixture, providing a continuous regeneration of ATP, no plateau was reached after 26 h of reaction time and during this time interval 99.6% of the hexokinase and ATPase activities had been lost (Fig. 1). Under these conditions the inactivation kinetic followed a first-order process until 90% of the enzyme activity (h = 9 x 10e3min-‘) was lost. Moreover, the native enzyme treated under the same conditions but in absence of ATP, lost only 5% of the initial activity. The incorporation of 32Pinto the protein was parallel to the loss of the enzyme activity. Moreover, the complete inactivation of the enzyme was obtained when one phosphate group was incorporated per enzyme subunit (molecular weight, 51,000) (insert Fig. 1). Reactivation
of the Modified
Enzyme
The inactivated enzyme (less than 1% activity) can be reactivated after gel filtration by incubation with ADPMg and Dxylose or n-lyxose (Fig. 2). The reactivation is in both cases a first-order process, nearly three times faster in the presence of xylose than in the presence of lyxose (h = 8.3 x 10e3 and 3 x 10m3min’, respectively). 32Pwas released from the protein
37
HEXOKINASE
in the presence of xylose at the same rate as the enzyme was reactivated (Fig. 2). The reactivation effect is quite specific. As shown in Table I, both the nucleotide (ADP) and the metal were essential requirements for the reactivation, as was the presence of one of the sugars cited above, xylose or lyxose. All other sugars assayed, whether they were substrates or products in the hexokinase reaction, were unable to induce the reactivation process. Moreover, the enzyme was not reactivated if AMP was substituted for ADP (Table I). On the other hand, when glucose was added to the reactivating system containing xylose and ADPMg, we observed an inhibitory effect on the enzyme reactivation rate (Fig. 3). Moreover, fly-CH,-ATP, an analog of ATP, has a similar inhibitory effect on the enzyme reactivation. The addition of P-y-CH,-ATP to the reactivating system at a concentration of 0.1 M with an equivalent addition of MgCl, reduces the rate of enzyme reactivation to 50% (results not shown). The 132Plphosphoenzyme also can be reactivated by incubation with alkaline TABLE
I
REACTIVATION OF THE PHOSPHOHEXOKINASE PRESENCE OF DIFFERENT LIGAND@
Additions
None Xylose Xylose and ADP Xylose, MgCl,, and ADP MgCl, and ADP Xylose, MgCl,, and AMP Lyxose Lyxose, MgCl,, and ADP Glucose Glucose, MgCl,, and ADP Glucosamme, MgCl,, and ADP Mannose, MgCl,, and ADP Fructose, MgCl,, and ADP Glucose-6-P, MgCl,, and ADP
IN THE
Recovered activity woo) 4.1 1.8 3.0 100.0 2.2 1.1 1.2 94.0 1.0 1.2 1.3 1.3 1.0 1.0
a After filtration through a Bio-Gel P-10 column the phosphoenzyme having 1% residual activity was incubated at 25°C in 25 mM tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer, pH 7, in the presence or absence of sugars (0.1 M), nucleotides (21 mM), and MgCl, (25 mM). The enzyme activity was assayed after 23 h of incubation. (For details, see Methods.)
38
MENEZES AND PUDLES
TIME
( min
)
FIG. 3. Inhibitory effect of glucose on the rate of phosphoenxyme reactivation. The phosphohexokinase was incubated at 25°C in the presence of 0.1 M xylose, 43 mM ADP, and 50 mM MgCl,. Glucose was added to a final concentration of 0.3 (0), 0.6 (A), 0.9 (O), 1.2 (O), or 1.5 (A) mM. A control was done without addition of glucose (W. In this figure, the straight lines represent the kinetics of the enzyme reactivation. A”, relative activity of the native enzyme; Al, relative activity of the phosphoenzyme at different time intervals.
phosphatase. Under these conditions, the [32P]phosphoenzyme was rapidly reactivated with a decrease in 32Pradioactivity of the phosphate-bound protein (Fig. 4). CONFORMATIONAL
STUDIES
Reactivity of the Sulfiydryl Groups of the Native and Phosphorylated Hexokinase Hexokinase A has four sulthydryl groups per subunit, one fast- and three slow-reacting groups, when titrated with Nbs, (12).2 Similar results were obtained for the phosphorylated enzyme, having 12% residual activity, after 6 h of incubation with xylose and ATPMg and elimination of the nucleotide by gel filtration through a Bio-Gel P-10 column previously equilibrated with 0.1 M xylose solution in 0.1 M Tris buffer, pH 8. As shown in Fig. 5, for both the native enzyme and the modified enzyme, the reaction of the sulfhydryl groups with Nbs, is biphasic, but the reaction rate of the three slow-reacting sulfhydry1 groups in the phosphoenzyme (k = 0.076 min-‘1 was enhanced by a factor of 3.5 as compared with the rate for the native enzyme (k = 0.022 min-‘). 2 Abbreviations used: Nbs,, 5,5’-dithiobis(2-nitrobenzoic acid); DEAE, diethylaminoethyl.
If the incubation with xylose and ATPMg was prolonged until the loss of 98% of the activity and the reactivity of the sulfhydryl groups were measured after elimination of the nucleotide and the sugar by gel filtration, the reactivity of the three “slow-reacting” sulfhydryl groups was found to be enhanced 10 times in the modified enzyme as compared to the native enzyme (see Table II). However, the addition of xylose or glucose significantly reduces the reactivity of the slow-reacting sulfhydryl groups (13) both in the native
‘Y’“”
TIME
(min)
FIG. 4. Reactivation of the phosphohexokinase by treatment with alkaline phosphatase. This figure represents both the recovery of the enzyme activity (W) and the release of 32P (0) from the 32P-labeled phosphoenzyme (0.25 mglml) incubated with alkaline phosphatase (1 mglml) at 3o”C, in 0.1 M Tris buffer, pH 7.6, containing 0.4 M NaCl.
30
60
90
120
160
180
TIME (min) FIG. 5. Determination of sulthydryl groups of the native and phosphoenzyme using Nbs, in the presence of xylose. Native enzyme (A); phosphoenzyme with 12% residual activity (0) (for experimental details, see Methods). The inset represents the semilogarithmic plot of the residual sulfhydryl groups versus time in the reaction with Nbs,.
PROPERTIES TABLE
OF PHOSPHORYLATED
II
RATE CONSTANTS OF THE “SLOW REACTING” SULFHYDRYL GROUP WITH Nbs, IN THE NATIVE HEXOKINASE AND IN THE PHOSPHOENZYMIF Rate constant (min-9 Additions
None D-Xylose (100 rnM) D-Glucose (5 m&f)
Native enzyme
Phosphoenzymeb
0.033 0.024 0.003
0.342 0.228 0.014
a Proteins were in 0.1 M Tris buffer, pH 8.0 and the reaction was carried out at the same pH. b Phosphoenzyme obtained after gel filtration and having 2% residual activity. (For details, see Methods.)
HEXOKINASE
39
sion spectra for the phosphorylated enzyme (not shown). From the chemical reactivity of the sulfhydryl groups of the phosphorylated hexokinase, there was an indication of a conformational change on the protein as compared with the native enzyme. However, using the fluorescence technique, we were unable to detect a significant perturbation of the protein structure due to the phosphorylation of the enzyme. It was thus important to determine whether a conformational change could be observed during the inactivation process by circular dichroism in the far ultraviolet region. By this method we could not observe any spectral difference between the native and phosphorylated enzyme, which showed a 94% loss of initial enzyme activity.
and in the phosphorylated enzyme. Indeed, as can be seen in Table II, the rate constant for the reaction of the sulfhydryl groups was reduced by a factor of 1.4 or 11 Binding of Glucose and ATP to the Native by the addition of xylose or glucose, respecand Phosphorylated Enzyme tively, to the native enzyme, and by a Zewe et al. (16) have shown that the factor of 1.5 or 24 if the same ligands were hexokinase fluorescence is quenched by added to the phosphorylated enzyme. the presence of glucose, ATP, and ATPMg. The dissociation constants that they obIntrinsic Fluorescence and Circular Ditained were similar to those obtained kichroism Studies of the Native and Phosnetically. Their results indicated that the phorylated Hexokinase fluorescence quenching was the result of The change in the reactivity of the binding of the substrates at the enzyme active site. sulfhydryl groups due to the inactivation process suggested that the conformation of the protein was affected by the phosphorylation of the essential serine residue. Since fluorescence methods have been found to be particularly sensitive to molecular changes in conformation of proteins (14, 15), it was interesting to observe whether, using this method, a conformational transformation of the protein in the course of the inactivation process could be detected. The kinetics of the inactivation process TIME ,mm, was followed by measurement of the loss of FIG. 6. Intrinsic fluorescence of hexokinase and the enzyme activity and by the change on the fluorescence emission at 330 nm (ex- loss in enzyme activity during incubation with xycited at 295 nm). As can be seen in Fig. 6, lose and ATPMg. The relative fluorescence intensity at 330 nm was measured during the incubation of only a slight decrease of fluorescence was hexokinase with xylose (0) or xylose and ATPMg detected during the reaction course as (0). Throughout the experiment, aliquots were compared with results for the native en- taken and assayed immediately for hexokinase aczyme under similar conditions but without tivity (A). F,,, fluorescence intensity at the time ATP addition. Moreover, we did not ob- zero; F, fluorescence intensity at different incubaserve any change in the maximum emis- tion times. (For experimental details, see Methods.)
40
MENEZES
AND
PUDLES
ciation constants for ATP with the binary complex were 0.55 and 1.74 mM, respectively, for the native and modified enzyme. Our dissociation constant values obtained with the native enzyme are somewhat higher than those observed by Zewe et al. (16). However, from our results it is evident that the substrates were capable of binding to the phosphoenzyme, though with a higher Kd. -4
-2
2
4
6
l/
8
10
I GLUCOSE]
12
I4
(mM-‘l
FIG. 7. Binding of glucose and ATP to the native and phosphoenzyme studied by fluorescence. (A) A double-reciprocal plot of the quenching effect of glucose on the enzyme fluorescence at 330 nm (excited at 295 nm) versus the glucose concentration. Native enzyme (A); phosphoenzyme (0) (see Methods). (B) A double-reciprocal plot of the quenching of ATP on the enzyme fluorescence at 330 nm (excited at 295 nm) versus ATP concentration. The enzyme was in the presence of 5 mM glucose. Native enzyme (A); phosphoenzyme (0).
We observe that the quenching effect which results from the binding of glucose is independent of the quenching effect induced by ATP. We were able to determine by the fluorescence method the binding constant for both glucose and ATP on the binary complex (enzyme-glucose). Figures 7A and B show double-reciprocal plots of the variation in the relative fluorescence intensity as a function of the ligand concentration. The dissociation constant were calculated from a linear regression analysis and the values found for glucose were 0.44 and 0.63 mM, respectively, whereas the disso-
DISCUSSION
In a preceding paper (4) we showed that the inactivation of yeast hexokinase induced by n-xylose and ATPMg is directly related to the phosphorylation of one serine residue per enzyme subunit. The phosphoenzyme, when incubated in the presence of xylose and ADPMg, can be dephosphorylated and reactivated with the same rate constant as that found for the inactivation process. Moreover, the same reactivation effect is also observed when the phosphoenzyme is incubated in the presence of lyxose and ADPMg. Except for the effect of alkaline phosphatase, which hydrolyzes the protein-bound phosphate and reactivates the phosphoenzyme, the inactivation and reactivtion processes are strictly dependent on the formation of a quaternary complex between the protein, xylose or lyxose, and ATPMg for the inactivation or ADPMg for the reactivation process. Sols et al. (17) as well as Rudolph and Fromm (18) have shown that lyxose and xylose are competitive inhibitors of the hexokinase activity. On the other hand, Anderson and Steitz (19) concluded from their low-resolution X-ray diffraction studies yeast hexokinase-substrate complexes that there is only one sugar binding site per enzyme subunit and that xylose in the presence of ADPMg binds to the same site as glucose. Moreover, DelaFuente observed that glucose inhibits the inactivation process by xylose and ATPMg, whereas in this paper we show that glucose or p-y-CH,-ATP inhibit the reactivation process. All of these data suggest that phosphorylation occurs at the level of the enzyme-active center region. Neverthe-
PROPERTIES
OF PHOSPHORYLATED
less, the nonreactivation of the phosphoenzyme by the sugar substrates in the absence or presence of ADPMg indicates that this modified form of the enzyme is not the controversial “phosphoryl-enzyme” intermediate of the hexokinase reaction (20). From the reactivation studies and fluorescence binding studies, it is quite clear that the substrate-binding sites on the enzyme do not seem to be significantly affected by the phosphorylation process. Even though the I-Cdvalues for glucose and ATP have been increased 1.2- and 3-fold, respectively, this increase could not explain the complete loss of the enzyme activity, since the enzyme assays were done with a substrate concentration much higher than the I& values, On the other hand, the conformational studies which were done by circular dichroism and fluorescence did not indicate any significant perturbation of the protein structure in the course of the phosphorylation. Jones et al. (21) and Otieno et al. (22), from their studies on the reactivity of the sulfhydryl groups of hexokinase B, suggested the possibility that some of the slow-reacting sulfhydryl groups could be in the proximity of the enzyme active center region. Then the observed change in the reactivity to Nbs, of the “slow reacting” sulfhydryl groups of the phosphoenzyme as compared with the native enzyme might quite possibly reflect a small perturbation in the vicinity of the enzyme-active center. However, this limited perturbation in the protein structure does not affect the formation of the quaternary complex of the phosphoenzyme with xylose or lyxose and ADPMg in the reactivation process, suggesting that the geometry of the enzymeactive site is preserved in the phosphohexokinase . The above results indicate that the reactive serine residue does not seem to be directly involved in the substrate binding sites or in the maintenance of the protein structure. Moreover, these results suggest that the phosphorylation of yeast hexokinase probably results from a specific conformational change of the enzyme active site induced by xylose or lyxose and
HEXOKINASE
41
ATPMg. This conformational change might modify the microenvironment of the serine residue, decreasing its PK, and consequently increasing its nucleophilicity, and furthermore, orienting the primary hydroxyl group into the vicinity of the yphosphate group of ATP. On the other hand, from the data described above we cannot exclude the possibility that this serine residue is implicated in the mechanism of action of yeast hexokinase. For the moment, we have no idea of the eventual physiological significance (if any) of this inactivating phosphorylation process; nonetheless, it represents an interesting and specific method for labeling an essential residue of hexokinase. ACKNOWLEDGMENTS This work was supported by grants from the Centre National de la Recherche Scientifique (Equipe de Recherche No. 142), Delegation Generale a la Recherche Scientitique et Technique (Contract No. 757.0188). We are grateful to Mrs. F. E. Tejtel and Dr. J. van Heijenoort for revising this manuscript. REFERENCES 1. DELAF’UENTE, G., LAGUNAS, R., AND SOLS, A. (1970) Eur. J. Biochem. 16, 226-233. 2. DELAFUENTE, G. (1970) Eur. J. Biochem. 16, 240-243. 3. CHENG, L. Y., INAGAMI, T., AND COLOWICK, S. P. (1973) Fed. Proc. 667, Abstract. 4. MENEZES, L. C., AND PUDLES, J. (1976) Eur. J. Biochem. 65, 41-47. 5. MENEZES, L. C. (1975) FEBS Meeting, Paris, Abstract 678. 6. RUSTUM, Y. M., RAMEL, A. H., AND BARNARD, E. A. (1971) Prep. Biochem. 1, 309-329. 7. HAMMES, G. G., AND KOCHAVI, D. (1962) J. Amer. Chem. Sot. 84, 2069-2073. 8. LAZARUS, N. R., RAMEL, A. H., RUSTUM, Y. R., AND BARNARD, E. A. (1966) Biochemistry 5, 4003-4016. 9. CLAUSEN, T. (1968)Anal. Biochem. 22,70-73. 10. DUBOIS, M., GILLES, K., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956)Anal. Chem. 28, 350-356. 11. ELLMANN, G. L. (1959) Arch. Biochem. 82, 7077. 12. ROSSI, A., MENEZES, L. C., AND PUDLES, J. (1975) Eur. J. Biochem. 59, 423-432. 13. GROUSELLE, M., (1969) Thesis “Doctorat de 3eme
42
MENEZES
Cycle,” Universitk de Paris, Centre d’Orsay. 14. ANDERSON, S., AND WEBER, G. (1966)Arch. Biothem. Biophys. 116, 207-223. 15. MENEZES, L. C., GROUSELLE, M., ANDPUDLES, J. (1972) Eur. J. Biochem. 30, 81-92. 16. ZEWE, V., FROMM, H. J., AND FABIANO, R. (1964) J. Biol. Chem. 239, 1625-1634. 17. Sons, A., DELAFUENTE, G., VILLAR-PALLASI, C., AND ASENSIO, G. (1958) Biochim. Biophys. Acta 30, 92-101. 18. RUDOLPH, F. B., AND FROMM, H. J. (1971) J.
AND
PUDLES
Biol. Chem. 246, 2104-2110. 19. ANDERSON, W. F., AND STEITZ, T. A. (1975) J. Mol. Biol. 92, 279-287. 20. WALSH, C. T., JR., AND SPECTOR, L. B. (1971) Arch. Biochem. Biophys. 145, l-5. 21. JONES, J. G., OTIENO, S., BARNARD, E. A., AND BHARGAVA, A. K. (1975) Biochemistry 14, 2396-2402. 22. OTIENO, S., BHARGAVA, A. K., BARNARD, E. A., AND RAMEL, A. H. (1975) Biochemistry 14, 2403-2410.
