Biochimica et BiophvsicaActa 831 (1985) 201-206 201 Elsevier

BBA32306

A f luorescence study of thermally induced conformational changes in

yeast hexok inase

Z y g m u n t Wasy lewski *, Nick L. Cr i sc imagna and Paul M. Horowi t z **

Department of Biochemistry, The University of Texas Health Science Center, 7703 Flqvd Curl Drive, San Antonio, TX 78284 (U.S.A.)

(Received May 29th, 1985)

Key words: Fluorescence; Hexokinase conformation: Thermal transition; (S. cerevisiae)

Fluorescence studies have been performed on yeast hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) as a function of temperature. Observations of both the intrinsic protein fluorescence and the fluorescence of the noncovalently bound apolar probe 2-(p-toluidinyl)naphthalene-6-suifonic acid under conditions where hexokinase is monomeric, indicate that significant thermal structural transitions occur in the protein over the physiological range of temperature (0°-40°C) and that there are different temperature- dependent forms of the enzyme. Thermal transitions between these forms are affected by the binding of the substrates D-glucose and ATP-Mg. It therefore appears that catalysis connects conformers that differ in stability and the present results are consistent with models in which hexokinase function is linked to changes in the interactions between the domains into which this protein is folded.

Introduction

Yeast hexokinase (ATP:D-hexose 6-phos- photransferase, EC 2.7.1.1) catalyses the transfer of the 7-phosphoryl group of ATP to D-glucose. The mechanism of the transfer involves the forma- tion of both a binary complex of the enzyme with glucose and a ternary complex involving enzyme, glucose and ATP. Many kinetic studies, under varying conditions of pH, the presence of sub- strates, etc., suggested that hexokinase undergoes a slow transition during the catalytic cycle [1-6]. Evidence from a variety of techniques such as changes in tryptophan fluorescence [1,7], dif- ference spectroscopy [7,8], low-angle X-ray scatter- ing [9] and fluorescence studies of the apolar probe

* Present address: Institute of Molecular Biology, Depart- ment of Biochemistry, Jagellonian University, Krakow, Po- land.

** To whom correspondence should be addressed.

2-(p-toluidinyl)naphthalene-6-sulfonic acid [10] suggest that hexokinase undergoes conformational changes in solution upon binding substrates.

X-ray diffraction studies have shown that hexokinase consists of two lobes separated by a deep cleft in which substrates are bound [11,12]. The enzyme crystallized as a complex with glucose has a conformation different from the conforma- tion of the enzyme crystallized in the absence of glucose [11]. Comparison of the high-resolution structures shows that in the presence of glucose one lobe of the molecule is rotated relative to the other lobe, resulting in closure of the interlobe cleft in which glucose is bound [11,12]. It has been suggested that in solution glucose can cause clos- ing of the cleft between the two lobes of hexokinase [12].

It is well known that the intrinsic fluorescence of proteins [13] as well as the fluorescence of noncovalently bound apolar probes [14] are very sensitive means of studying protein structures and

0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

202

changes therein. It is known that the temperature- dependence of the fluorescence properties of pro- teins in the physiological range of temperature is determined by the thermal dependence of the dy- namic mobility of protein structure surrounding the fluorophore [15]. The changes in thermal fluo- rescence quenching curves under varying condi- tions of substrates should express the conforma- tional flexibility of enzymes containing bilobed structures [16].

In fact, thermal transitions, which depend on substrate binding, are found in other proteins such as thiosulfate sulfurtransferase (thiosulfate : cyanide sulfurtransferase, EC 2.8.1.1) [16]. This enzyme, just like yeast hexokinase, consists of a single polypeptide chain folded into two lobes separated by a deep cleft in which substrates are bound [17]. Preliminary N M R experiments that required protein concentrations of 50 m g / m l have detected a thermal transition in hexokinase [18].

In the present study of yeast hexokinase, the temperature-dependence of the intrinsic tryp- tophanyl fluorescence, as well as the fluorescence of the noncovalently bound apolar probe 2-(p- toluidinyl)naphthalene-6-sulfonic acid were mea- sured under varying conditions of substrate con- centration, pH and ionic strength, and we report the presence of significant thermal transitions in the protein in the physiological range of tempera- ture.

Materials and Methods

Crystalline yeast hexokinase was purchased from Sigma Chemical Company (No. H-5750) as an ammonium sulfate suspension and is almost entirely the PI isomer. The enzyme was dialyzed against 0.01 M Tris-HCl buffer (pH 7.5) or against 0.01 M Tris-HCl, (pH 8.0)/0.1 M KC1 then centrifuged prior to fluorescence measurements. Protein concentration was determined from ab- sorbance measurements and the E~8~ was assumed to be 9.47 [19].

fl-D-Glucose was obtained from Nutritional Biochemical Company (Cleveland, OH) and ATP disodium salt was purchased from Boehringer- M a n n h e i m , F . R . G . 2 - ( p - T o l u i d i n y l ) - napththalene-6-sulfonic acid was purchased from Molecul~ir Probes (Junction City, OR). The 2-(p-

toluidinyl)naphthalene-6-sulfonic acid concentra- tion was determined from absorbance measure- ments at 317 nm and the molar extinction coeffi- cient was assumed to be 18 900 [20]. Other chem- icals used were reagent grade.

Intrinsic fluorescence emission spectra were re- corded with an SLM spectrofluorimeter ( S L M / Aminco, Urbana, IL). Excitation was 295 nm and spectra were recorded from 300 to 400 nm. The fluorescence spectra of hexokinase containing 2- ( p-toluidinyl)naphthalene-6-sulfonic acid at a con- centration of 50/~M were obtained with excitation at 366 nm and recorded from 370 to 470 nm. In each measurement of the fluorescence emission spectrum the enzyme concentration was 0.2 m g / ml. In the enzyme solutions containing substrates the D-glucose concentration was 20 mM and both ATP and MgC1 z were equal to 0.1 mM. To pro- duce an equilibrium mixture containing all rele- vant enzyme intermediates, hexokinase was prein- cubated with the mixed substrates at these con- centrations and preincubated for 30 min at 25°C. Fluorescence measurements were performed as a function of time under a single set of environmen- tal conditions to ensure that no further change in the observed parameters would occur.

Intrinsic fluorescence lifetimes were measured on a phase-modulation cross-correlation SLM flu- orimeter and all reported values are from phase lifetime measurements obtained by modulation at 30 MHz with excitation at 295 nm. For hexokinase, with multiple tryptophans, the measured lifetime is an average value. In each experiment the enzyme concentration was 0.4 m g / m l . In the enzyme solu- tion containing ligands the D-glucose concentra- tion was 40 mM and the concentrations of both ATP and MgC12 were equal to 0.2 mM. An equi- librium mixture was generated as above.

The cuvette temperature was controlled by cir- culating water, and fluorescence measurements were made following each temperature change after waiting 10 min for thermal equilibration.

Ultracentrifugation was performed using a Be- ckman Model E analytical ultracentrifuge equipped with Schlieren optics. Samples of hexokinase were sedimented in a double-sector cell at 47251 rpm for 90 min at a temperature of 25°C and a hexokinase concentration of 2 m g / m l in a buffer consisting of 0.01 M Tris-HC1 (pH 7.4). Glucose

was included at 20 mM to assess its effect on the sedimentation behavior of the enzyme. Photo- graphs were taken every 8 min during sedimenta- tion and the rate of boundary movement was used to calculate s20.w values.

Gel-filtration chromatography was performed on a column containing AcA-44 (LKB) (0.9 × 120 cm) and developed at a rate of 4.4 m l / h using a buffer consisting of 0.01 M Tris-HC1 (pH 7.5) either alone or supplemented with 2 mM glucose. The column was calibrated with the following proteins: cytochrome c, chymotrypsinogen, ovalbumin, bovine serum albumin and aldolase. The column was calibrated for each buffer condi- tion used.

Results

Intrinsic fluorescence properties were measured for yeast hexokinase alone and with various com- binations of its substrates. Fluorescence was ex- cited at 295 nm to minimize excitation of tyrosine [13]. The fluorescence emission maximum occurred at 331 nm with and without the substrates, ATP- Mg and D-glucose up to about 10°C (Figs. 1 and

I0

> , 8

~'e= 6

8 ~ 4

~ 2

A

I I 1 I I

z a

i 3401 L; do s'o 4'0 do

Temperature (%) Fig. 1. Temperature-dependence of theintrinsic fluorescence properties of yeast hexokinase in 0.01 M Tris-HCl buffer (pH 7.5) when excited at 295 rim. (A) Fluorescence intensity mea- sured at the max imum of the fluorescence emission. (B) Wave- length of the max imum of the fluorescence emission. ©, Hexokinase alone; e , hexokinase in the presence of 20 m M of glucose. Protein concentration was 0.2 mg / ml .

203

~ c

g~

~N 2 -

kl_

O- ~ J ' l J B~ E 2 s40 I

E-- = E 330 ~ ~t~-~°

0 c

. _ ~

E 320~ ~ • " ' . , ,o do s'o 4o go o

Temperature (%)

Fig. 2. Temperature-dependence of the intrinsic fluorescence properties of yeast hexokinase in 0.01 M Tris-HC1 buffer (pH 7.5). O, Hexokinase in the presence of 0.1 mM of both ATP and MgCI2; e, hexokinase in the presence of 20 mM glucose and of 0.1 mM of both ATP and MgCI 2. Other denotations the same as in Fig. 1.

2), consistent with the idea that the tryptophan residues of hexokinase are buried [21]. However, the fluorescence intensity of the enzyme contain- ing glucose was about 15% lower than the fluores- cence intensity in the absence of this substrate, consistent with previous reports [22-24].

In the absence of glucose, the slopes of curves showing the temperature dependence of the fluo- rescence intensity and the emission maximum change abruptly at approx. 30°C (Fig. 1), and suggest a thermal transition which occurs in the protein molecule in the physiological range of temperature. Upon binding D-glucose, hexokinase shows a different thermal fluorescence-quenching curve and temperature transitions are observed at 40°C.

When the complex between ATP-Mg and hexokinase occurs without D-glucose, the thermal fluorescence quenching shows an additional transi- tion in the enzyme which occurs at about 16°C (Fig. 2A), without significant change in the maxi- mum of the fluorescence spectrum (Fig. 2B). In contrast, the equilibrium mixture does not display the thermal transition at 16°C shown in Fig. 2A. However, in the equilibrium mixture the maximum of the fluorescence emission was shifted about 2 nm to lower wavelength (Fig. 2B).

The plots of the reciprocal of the intrinsic fluo-

204

Temperature (°C) . 0 Io 20 so 40 so 6o 7o

i

_~os r

02 l ~ I 2 3 4 5 6 8

T/i'~.× 10 .4

Fig. 3. The dependence of the reciprocal of the intrinsic fluores- cence lifetime on T/B when excited at 295 nm in 0.01 M Tris-HCI buffer (pH 7.5). O, Hexokinase alone; e , hexokinase in the presence of 40 mM glucose. Protein concentration was 0.4 mg/ml.

rescence lifetimes versus T / ~ for hexokinase in the presence and absence of D-glucose in 0.01 M Tris- HCI buffer (pH 7.5) are presented in Fig. 3. With hexokinase alone, 1/~- vs. T/~I is a linear function up to about 36°C, where a thermal transition occurs. At higher temperature, in the range from about 45°C to 57°C, another thermal transition is observed. In the presence of D-glucose the enzyme displays the thermal transition at higher tempera- ture as shown in Fig. 3. However, it should be pointed out that at a lower temperature (less than 300C), the hexokinase-b-glucose complex is char- acterized by a lower intrinsic fluorescence lifetime value and a different slope of the 1 /7 vs. T / ~ (Fig. 3) ~ as compared to the enzyme alone and this may reflect a different conformational state in- duced by the addition of glucose.

Ultracentrifugation and gel-filtration experi- ments demonstrated that hexokinase remained monomeric during the present experiments: Ultra- centrifugation gave S2o., , = 4.2 S in the absence of glucose, and S2o 2 = 4.1 S in the presence of glu- cose, consistent with monomeric hexokinase (M r = 51000). Further, gel filtration on a calibrated AcA-44 column yielded a Stokes radius of 33 for hexokinase, independent of whether glucose was present.

2-(p-Toluidinyl)naphthalene-6-sulfonic acid is one of a class of compounds that are weakly

8 r , - - •

E'u~ 6

LI . . I L ~ I . ~ I

E 440~

F=- so t -~ 420

E w 4106 Io 20 3'0 4b sb

Temperature (°C)

I i

J 6O

Fig. 4. Temperature-dependence of the 2-(p-toluidinyl)naph- thalene-6-sulfonic acid binding properties of yeast hexokinase in 0.01 M Tris-HC1 buffer (pH 7.5), when excited at 366 nm. O, Hexokinase alone; e, hexokinase in the presence of 20 mM glucose. (A) Fluorescence intensity measured at the maximum of the fluorescence emisison. (B) wavelength of the maximum of the fluorescence emission. Protein concentration was 0.2 mg /ml and 2-(p-toluidinyl)naphthalene-6-sulfonic acid con- centration was 5.10 s M.

fluorescent in water but become highly fluorescent upon binding to apolar sites on proteins [14]. When D-glucose is bound to hexokinase at 20°C in the presence of 2-(p-toluidinyl)naphthalene-6- sulfonic acid, then its fluorescence intensity in- creases and the fluorescence maximum is shifted to longer wavelengths (Fig. 4). This result is con- sistent with those reported previously [10]. These observations suggest that hexokinase has apolar binding sites and that there is a change in solvent accessible apolarity when glucose is bound a n d / o r that binding leads to a more rigid environment for the probe.

The temperature dependence of the 2-(p- toluidinyl)naphthalene-6-sulfonic acid fluores- cence properties shown in Fig. 4 reveal sharp transitions occurring at about 17°C in hexokinase alone and at 29°C in the enzyme-glucose complex. With further temperature increases, new transi- tions are observed at about 36°C and 40°C as monitored by the increase of 2-( p-toluidinyl)naph- thalene-6-sulfonic acid fluorescence intensity with both hexokinase alone and in the presence of glucose, respectively. Similar temperature-depen-

dences of the 2-(p-toluidinyl)naphthalene-6- sulfonic acid fluorescence properties were detected for the complex of hexokinase-ATP and the hexokinase equilibrium mixture (data not shown) as were observed with hexokinase alone and in the presence of glucose (Fig. 4). However, for the enzyme-ATP complex both temperature-induced transitions were shifted slightly, to about 15°C and 32°C, respectively.

Discussion

The thermal transitions detected inthis study may reflect the dependence of the functional prop- erties of yeast hexokinase on the dynamic behavior of the enzyme under a variety of conditions such as the presence and absence of substrates and variations of temperature. There are potential am- biguities in estimates of transitions in proteins with multiple tryptophans where the thermal re- sponse is not clearly composed of intersecting straight lines. However, when closely related con- formers are being compared, estimates of transi- tion temperatures can give valuable relative infor- mation. Hexokinase is important for this study, since the conformers in question are related by the binding of substrates that introduce no optical ambiguities. In addition, the thermal behavior can be independently monitored using the apolar probe 2-(p-toluidinyl)naphthalene-6-sulfonic acid, thus further reducing interpretive uncertainties.

The results of measurements of intrinsic fluo- rescence as well as the fluorescence of the nonco- valently bound apolar probe 2-( p-toluidinyl)naph- thalene-6-sulfonic acid, indicate that in solution yeast hexokinase undergoes a conformational change upon binding glucose such that the result- ing complex has increased stability. These results are in agreement with previously published evi- dence obtained by low-angle X-ray scattering tech- niques [9], ultraviolet difference spectroscopy [7,8] and intrinsic fluorescence spectroscopy .[7].

The results also indicate that up to about 16°C the enzyme-ATP complex exists in a conformation slightly different from hexokinase alone. This con- formational state is characterized by a different thermal fluorescence quenching process and may result from a small reorientation of one of the enzyme domains relative to the other. This sugges-

205

tion is supported by the low-angle X-ray scattering measurements from solutions of yeast phospho- glycerate kinase in the presence of ATP-Mg, in which a small reduction (0.58_+ 0.32 A) in the radius of gyration of the enzyme was detected upon binding nucleotide [25]. Since this different conformational state of hexokinase-ATP is pro- duced as a result of a low-temperature structural transition, it is expected that the necessary energy required for this conformational change is rather small.

The thermal transitions, which occur in the physiological range of temperature in yeast hexokinase are very similar to the thermal transi- tions, which are found in other proteins such as thiosulfate sulfurtransferase [15]. This enzyme also consists of two lobes separated by a deep cleft in which substrates are bound [17]. In addition, it should be noted that the present results are con- sistent with recent studies of hexokinase at high concentrations by N M R [18] and by the binding of TNS at 25°C [10], which gave results that were also interpreted in terms of substrate-induced al- terations in the interactions between hexokinase domains.

It is therefore tempting to speculate that the thermal transitions observed here are related to changes in the interactions between the two do- mains into which hexokinase is folded. This would be in keeping with suggestions that hexokinase exists in an 'open ' conformation that 'closes' to varying degrees when substrates are bound.

Acknowledgements

This research was supported by grant GM25177 form the National Institutes of Health and grant AQ 723 from the Robert A. Welch Foundation.

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