Bioscience Reports, Vol. 20, No. 1, 2000
Increased Stability and Catalytic Efficiency of Yeast Hexokinase Upon Interaction with Zwitterionic Micelles. Kinetics and Conformational Studies Rodrigo Guerra1 and M. Lucia Bianconi2,3 Received October 15, 1999; accepted November 2, 1999 The effect of ligands (glucose, ATP and Mg2+) and zwitterionic micelles of lysophosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl-3-ammonium propanesulfonate (HPS) in the yeast hexokinase (HK) stability was studied at 35°C. The thermal inactivation kinetics followed one-exponential decay. The effect of ligands on protecting the enzyme against inactivation followed the order: glucoseHglucoseyMg2+HATPyMg2+ >Mg2+ >buffer only. Both LPC and HPS micelles increased the enzyme stability only when the incubation medium contained glucose or glucoseyMg2+, suggesting that the protein conformation is a key prerequisite for the enzyme-micelle interaction to take place. This enzyme-micelle interaction resulted in an increased catalytic efficiency (with a decrease in Km for ATP and increase in Vmax as well as in changes on the tertiary (intrinsic fluorescence) structure of the yeast hexokinase. KEYWORDS: Yeast hexokinase; Stability; Enzyme-micelle interaction; Fluorescence; Zwitterionic micelle. ABBREVIATIONS: HK, hexokinase; HPS, N-hexadecyl-N,N-dimethyl-3-ammonium propanesulfonate; K, glucose dissociation constant; LPC, lyso-phosphatidylcholine; Tris,tris (hydroxymethyl) aminomethane.
INTRODUCTION In biotechnology, it is very important to find new ways of increasing the stability of enzymes that usually are very liable and can denature under various experimental conditions. Therefore, the use of microheterogeneous media, such as reversed micelles, to improve the enzyme performance has been subject of attention for several years [1–11]. The study of different properties of enzymes when solubilized in reversed micelles is also important to understand the properties of water-soluble enzymes in a system that resembles the in vivo situation where enzymatic reactions may occur in the proximity of interfaces [5]. Furthermore, the water inside of a 1
Departamento de Bioquı´mica Me´dica, ICByCCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, CEP 21941-590, Brazil. 2 Departamento de Bioquı´mica Me´dica, ICByUFRJ, Pre´dio do CCS, bloco E, sala 38, Ilha do Funda˜o, Rio de Janeiro, RJ, CEP 21941-590, Brazil. 3 To whom correspondence should be addressed: e-mail:
[email protected] 41 0144-8463y00y0200-0041$18.00y0 2000 Plenum Publishing Corporation
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living cell does not behave ideally as it is solvating and interacting with different macromolecular assemblies, organic molecules or inorganic ions. From this point of view, the use of model systems such as micelles, can contribute to understand the function of enzymes in their natural environment. Hexokinase (ATP :D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) is a key enzyme in the glycolysis which catalyses the phosphorylation of glucose to glucose6-phosphate. It is a dimeric protein consisting of two identical subunits of 52 kDa [12–14]. The conformation of each subunit consists of two lobes separated by a deep cleft where glucose binds. Binding of glucose induces a great conformational change in which one lobe rotates 12° relative to the other lobe, closing the cleft [15] and resulting in an increased affinity for ATP [16, 17]. Here we show that both the thermal stability and catalytic efficiency of the yeast hexokinase increases upon interaction with zwitterionic micelles of lyso-phosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl propane sulfonate (HPS). This enzyme-interface interaction occurs only when glucose is bound to the enzyme, suggesting that the conformation change induced by the ligand [15] is the key prerequisite for the interaction to take place. As a result of the interaction, there is a conformational change as observed by the red-shift (from 330 to 340 nm) in the intrinsic fluorescence spectrum of the enzyme. METHODS Assay for Hexokinase Activity The activity of yeast HK (Sigma) was determined at room temperature by measuring the formation of NAD(P)H at 340 nm in the coupled reaction with Glucose6-phosphate dehydrogenase from Leuconostoc mesenteriodes (G6PD; EC 1.1.1.49; Sigma). The assay medium contained 50 mM Tris :HCl pH 7.5, 10 mM glucose, 5 mM MgCl2 , 1.2 mM ATP, 1.0 mM NAD(P)+, and G6PD (0.4 Uyml). The reaction was started by addition of one or more components absent in the incubation medium, and followed for at least 3.5 min. The reaction volume was 500 µl. All reagents were analytical grade. Thermal Stability of Yeast Hexokinase The yeast HK (0.1 Uyml) was incubated in five different media (see below) at 35°C in the absence or in the presence of zwitterionic micelles (0.5 mgyml). After equilibration of the incubation medium at 35°C, the enzyme was added and, at regular time intervals, samples (500 µl) were withdrawn in order to measure the enzyme activity as described above. The residual HK activity was calculated as a percentage of the original activity (considered 100%), obtained at tG0 min incubation. The incubation media prepared in 50 mM Tris :HCl buffer, pH 7.5 were: (i) buffer only, or buffer containing (ii) 10 mM glucose, (iii) 5 mM MgCl2 , (iv) both 10 mM glucose and 5 mM MgCl2 , or (v) 1.2 mM ATP and 5 mM MgCl2 . Aqueous micelles of lyso-phosphatidylcholine (LPC; Sigma) or N-hexadecylN,N-dimethyl-3-ammonium propanesulfonate (HPS; Sigma) were obtained by dissolution of the surfactant in the incubation medium and pre-incubated at 35°C
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before addition of the enzyme. The surfactant concentration was 0.5 mgyml. The same procedure was done for the determination of the HK stability in the presence of micelles. Binding of Glucose The binding of glucose to the yeast HK was studied by measuring the quenching of the intrinsic fluorescence of the protein upon binding of glucose [18]. Fluorescence measurements were carried out in a Hitachi F-3010 spectrofluorimeter. The excitation and emission wavelengths were 285 nm and 330 nm, respectively, with bandwidths of 5 nm. Titrations were done by adding small aliquots (3 to 5 µl) of glucose to the sample (1.5 ml) containing the yeast HK in 50 mM Tris :HCl, pH 7.5, either in the presence and in the absence of micelles (0.5 mgyml). The effect of dilution was corrected by adding same amounts of Milli-Q water to the enzyme solution. The HK concentration was 25 µgyml. The glucose dissociation constants (K) were obtained by applying the Eq. (1), [18] assuming that the binding occurs as a simple mechanism: 1 ∆f
G
1 K 1 C ∆fs ∆fs [G]
(1)
where ∆f is the fluorescence quenching at a given concentration of free glucose ([G]), and ∆fs is the maximum quenching obtained at saturating concentrations of glucose. Fluorescence Studies The HK intrinsic fluorescent spectra (from 310 to 400 nm) were obtained in different incubation times of the protein at 35°C in a Hitachi F-3010 spectrofluorimeter equipped with temperature control device. The excitation wavelength was 295 nm with bandwidths of 5 nm. The incubation medium contained 50 mM Tris :HCl, pH 7.5, 10 mM glucose and 5 mM MgCl2 , either in the presence and in the absence of LPC or HPS micelles (0.5 mgyml). The yeast HK concentration was 10 µgyml. RESULTS AND DISCUSSION Effect of Ligands and Zwitterionic Micelles on Yeast Hexokinase Stability at 35°C The effect of ligands (glucose, ATP and Mg2+) in the yeast HK stability at 35°C is shown in Fig. 1. The experimental curves were analyzed according to a monoexponential decay, allowing the calculation of the half-time for thermal inactivation (t0.5 , Table 1). Glucose was the only ligand that effectively increased the yeast HK stability with at least 10-fold increase in t0.5 (Table 1). The stabilizing effect of glucose on yeast hexokinase was already shown by calorimetric studies [19, 20] where the thermally induced transition occurs at higher temperature and δH than those observed in the absence of glucose. However, if along with glucose Mg2+ was present in the
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Fig. 1. Effect of ligands on yeast hexokinase stability at 35°C. The enzyme (0.1 Uyml) was incubated at 35°C, and the residual activity was measured at regular time intervals as described in Methods. The incubation media prepared in 50 mM Tris :HCl, pH 7.5 were: buffer only (n), or buffer containing 5 mM MgCl2 (&), 1.2 mM ATP and 5 mM MgCl2 (■), 10 mM glucose (s) or 10 mM glucose and 5 mM MgCl2 (●). The residual activity at different incubation times (means J S.E., nG5) was calculated as a percentage of the original activity, obtained at tG0 min incubation.
Table 1. Effect of Ligands and Zwitterionic Micelles on the t0.5 for the Thermal Inactivation of Yeast Hexokinase at 35°C t0.5 (min)a Ligand
No micelles
None Mg2+ ATPyMg2+ GlucoseyMg2+ Glucose
34.6 39.9 43.2 106.5 400.0
a
With LPC 33.7 40.8 60.0 638 n.d.b
With HPS 30.2 37.0 64.0 546 n.d.b
t0.5 is the time (in min) to reach 50% of the residual activity of the yeast hexokinase; it was calculated from the A1 , t1 , and y0 parameters obtained by the curve fitting of the experimental data in Fig. 1 and Fig. 2 as one-exponential decay. b n.d.—not determined; in both cases, the data could not be fitted as one-exponential decay due to the small change in the enzyme activity after 200 min incubation with the micelles. The calculation of t0.5 would not be accurate for this situation.
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incubation medium, although the enzyme remained more stable than in buffer only, the t0.5 for inactivation decreased when compared to that found with glucose. This result may be reflecting the findings reported by Shill et al. [21] in studies of the monomer–dimer equilibria of yeast HK applying the reacting enzyme sedimentation method. Glucose binding is known to induce the dissociation of the dimer [12, 13, 18, 21]. However, Shill et al. [21] showed that in the presence of glucose, the addition of MgCl2 caused an increase in the sedimentation velocity of the HK to values close to those obtained with the dimer. These authors suggested that MgCl2 could cause a conformational change in the enzyme to the original one observed in buffer alone or else, to a new conformation with sedimentation properties similar to that found for the dimer [21]. Therefore, the decrease in the HK stability observed with glucose and MgCl2 can be a result of a different enzyme conformation than that found with glucose alone. No significant increase in the enzyme stability was observed with Mg2+ or ATPy 2+ Mg in the incubation medium (Fig. 1 and Table 1). The results found with glucose alone or ATPyMg2+ were very similar to those obtained by Williams and Jones [22] in studies of the stability of this enzyme at 30°C. In the glucose-containing media, the addition of LPC or HPS micelles causes a significant increase in the HK stability at 35°C (Fig. 2, and Table 1) indicating that the enzyme is able to interact with the micelle interface. This effect is more pronounced in the incubation with glucose and Mg2+, where the enzyme held around 80% of its original activity after 200 min incubation with micelles, while only 12% of the original activity was observed in the absence of micelles (Fig. 2B). On the other hand, when the yeast HK was incubated with LPC or HPS micelles in the absence of glucose, the protective effect against thermal inactivation of the enzyme is no longer observed (Table 1). It is well known that glucose binding induces a great conformational change in the enzyme [15]. Our results suggest that this conformational change favors the interaction of the enzyme with zwitterionic micelles, probably by exposing (or hiding) residues that allow (or restrain) the contact of the enzyme with the micelle interface. Effect of Zwitterionic Micelles on Km for ATP, Vmax , and Glucose Binding The consequences of the enzyme-interface interaction on the kinetic parameters (apparent Km for ATP and Vmax) of yeast HK were studied at room temperature by measuring the steady-state HK activity with ATP concentrations ranging from 0.04 to 1 mM, in the presence of saturating glucose and Mg2+ concentrations. It was observed that both HPS or LPC micelles cause a decrease in at least three times in the apparent Km for ATP (Table 2). This may be due to a local increase in the ATP concentration at the micelle interface. In addition, there was a twofold increase in the Vmax observed in the presence of micelles (Table 2). Consequently, the VmaxyKm ratio increased 5 times (Table 2) showing that the interaction of yeast HK with zwitterionic micelles leads to an increased enzyme efficiency. However, only a small change in the dissociation constant for glucose binding (K) was observed in the presence of LPC or HPS micelles. The titration curves
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Fig. 2. Effect of zwitterionic micelles on yeast hexokinase stability at 35°C. The enzyme (0.1 Uyml) was incubated at 35°C in a medium containing LPC or HPS micelles, and the residual activity was measured at regular time intervals as described in Methods. The incubation medium was prepared in 50 mM Tris :HCl, pH 7.5 containing 10 mM glucose (A) or 10 mM glucose and 5 mM MgCl2 (B), in the presence of LPC (s) or HPS (n) micelles. The micelle concentration was 0.5 mgyml (approximately 1 mM). The residual activity at different incubation times (means J S.E., nG5) was calculated as a percentage of the original activity obtained at tG0 min incubation. Data in the absence of micelles (●) are from Fig. 1.
Table 2. Effect of Zwitterionic Micelles on Km for ATP, Vmax , and in the Dissociation Constant (K) for Glucose Binding to Yeast Hexokinasea,b Condition No micelles With LPC With HPS a
Km for ATP ( µM)
Vmax ( µmol G6P min−1)
Vmax yKm
K for glucose (mM)
210J20 72J6 76J7
141J10 273J15 281J11
0.67 3.79 3.70
1.21 0.94 0.93
The Km for ATP was determined at room temperature from the steady-state HK kinetics with ATP concentrations ranging from 0.04 to 1 mM. Km and Vmax were calculated by fitting the Lineweaver–Burke plots of HK activity as a function of initial ATP concentration according to Michaelis–Menten kinetics. The HK activity was measured in the coupled reaction with glucose-6-phosphate dehydrogenase (see Methods for details). The values represent the means J S.E. (nG6). b K was determined from the quenching of the intrinsic HK fluorescence upon glucose binding, by fitting the plots of 1y∆fs as a function of 1y[G] according to Eq. (1) (see Methods), were rG0.995, 0.998, and 0.997 for the data in the absence of micelles, with LPC and with HPS, respectively.
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obtained by the quenching of the intrinsic protein fluorescence upon glucose binding were analyzed according to a hyperbolic function. Our results were very similar to those obtained by Hoggett and Kellett [18] at pH 7.5 with small HK concentration (8 µgyml). The plots of ∆fs as a function of 1y[G] were linear in all cases suggesting that the binding of glucose to yeast HK in the presence of micelles can be described by a single equilibrium mechanism. Similar values of K were found in aqueous medium or with LPC or HPS micelles (Table 2). This is probably due to the lack of
Fig. 3. Effect of zwitterionic micelles on the intrinsic fluorescence spectrum of yeast HK. The intrinsic fluorescence spectra of yeast HK were obtained at different incubation times of the enzyme in 50 mM Tris :HCl, pH 7.5, 10 mM glucose and 5 mM MgCl2 at 35°C. The effect of incubation at 35°C in the intensity (A) and in the λ max (B) of the intrinsic fluorescence spectrum of yeast hexokinase was studied in the absence (●) and in the presence of LPC (s) or HPS (n) micelles. The excitation wavelength was 295 nm, with excitation and emission bandpass of 5 nm.
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enzyme-micelle interaction in the absence of glucose, already suggested from the studies of thermal stability. Fluorescence Studies The emission maximum (λ max) of the intrinsic fluorescence spectrum of yeast HK in aqueous medium is 329 nm, indicating that the Trp residues are buried. When the enzyme was incubated at 35°C in aqueous medium containing glucose and Mg2+, there was a decrease in the intensity of the intrinsic HK fluorescence spectrum (Fig. 3A), although no change in emission maximum was observed (Fig. 3B). However, when LPC or HPS micelles were present in similar conditions, while no significant change in the fluorescence intensity occurs (Fig. 3A), there was a red-shift in the HK spectrum (Fig. 3B) where the emission maximum changed from 329 to 334 nm. This result suggests that a small change in the enzyme tertiary structure results from the interaction of yeast HK with the interface, as the Trp residues become more exposed to a polar medium. According to our findings, upon binding to zwitterionic micelles the yeast HK assumes a new, more stable, conformation. This new conformation of the enzyme is more resistant against thermal inactivation at 35°C and have an increased catalytic efficiency. ACKNOWLEDGEMENTS This work was funded by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and from the Programa de Apoio ao Desenvolvimento Cientı´fico e Tecnolo´gico (PADCT-CNPq). R.G. was recipient of fellowships from CNPq. REFERENCES 1. Martinek, K., Levashov, A. V., Klyachko, N. L., Pantin, V. I., and Berezin, I. V. (1981) Biochim. Biophys. Acta 657:277–294. 2. Fletcher, P. D. I., Rees, G. D., Robinson, B. H., and Freedman, R. B. (1985) Biochim. Biophys. Acta 832:204–214. 3. Martinek, K., Levashov, A. V., Klyachko, N. L., Khmelnitsky, Yu. L., and Berezin, I. V. (1986) Eur. J. Biochem. 155:453–468. 4. Luisi, P. L., Giomini, M., Pileni, M. P., and Robinson, B. H. (1988) Biochim. Biophys. Acta 947: 209–246. 5. Martinek, K., Klyachko, N. L., Kabanov, A. V., Khmelnitsky, Yu. L., and Levashov, A. V. (1989) Biochim. Biophys. Acta 981:161–172. 6. Fletcher, P. D. I., Freedman, R. B., Robinson, B. H., and Rees, G. D. (1987) Biochim. Biophys. Acta 912:278–282. 7. Ruckenstein, E., and Karpe, P. (1991) J. Phys. Chem. 92:6028–6032. 8. Karpe, P., and Ruckenstein, E. (1991) J. Colloid Interface Sci. 141:534–552. 9. Gajjar, L., Dubey, R. S., and Srivastava, R. C. (1994) Appl. Biochem. Biotechnol. 49:101–112. 10. Lalitha, J., and Mulamani, V. H. (1997) Biochem. Mol. Biol. Int. 41:797–803. 11. Rariy, R. V., Bec, N., Klyachko, N. L., Levashov, A. V., and Balny, C. (1998) Biotechnol. Bioeng. 57:552–556. 12. Easterby, J. S., and Rosemeyer, M. A. (1972) Eur. J. Biochem. 28:241–252.
Stabilization of Hexokinase 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
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