Biochimica et Biophysica Acla, 1120 (1992) 305-307

© 1992 Elsevier Science Piiblishers B.V. All rights reserved 016%4838/92/$05.00

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Effect of dimethyl sulfoxide on phosphoryl transfer catalyzed by yeast hexokinase Mdnica Montero-Lomeli lnstituto de Ciencias Biomedicas, Departamento de Bioquimica, Unicersidade Federal do Rio de Janeiro, Cemro de Ciencias da Saude, Cidade Unirersitaria. Rio de Janeiro (Brazil)

(Received 2 September 1991) (Revised manuscript 18 November 19tJl)

Key words: Hexokinas¢: Dimethyl sulfoxide; ATP hydrolysis Hexokinase is a phosphotransferase that catalyzes phosphoryl transfer from ATP to glucose much more rapidly than the transfer from A T P to water (i.e.. hydrolysis) Dimcthyl sulfoxide has opposite effects on these two phosphotransferase activities: it enhances ATP hydrolysis and inhibits glucose phosphorylation. Xylose, a sugar that is non-phosphorylatable by hexokinase, enhances ATPasc activity which is additive to activation by dimethyl suifoxide, indicating that the mechanism of activation by dimcthyl sulfoxide is different from that of xTIose. These results suggest that it is possible to change the specificity of the enzyme in the presence of dimethyl sulfoxide.


Yeast hexokinase is a water-soluble protein that catalyzes the transfer of a phosphoryl group from ATP to glucose much more efficiently than it hydrolyzes ATP. Evidence from several studies [1-4] suggests that hexokinase undergoes a conformational change upon glucose binding that may enhance the phosphoryl transfer to glucose. In recent years attention has been focused on changes in the specificity of enzymatic reactions caused by organic solvents. It has been shown that lowering the water activity of the medium with organic solvents such as dimethyl sulfoxide, change the catalytic properties of several ATPases, including the membrane-bound Ca2+-ATPase of sarcoplasmic reticulum. The presence of dimethyl sulfoxide increases the affinty of this enzyme for Pi, and the hydrolysis of ATP is impaired [5,6]. In this report, we examined the effect of dimethyl sulfoxide on yeast hexokinase, an enzyme that normally acts in an aqueous environment. The data presented here show that dimethyl sulfoxide changes the specificity of hexokinase, enhancing the ATPase activity,

Correspondence: M. Monteru-i_~ael[, Instituto de Ciencias Biomedleas, Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude, Cidade Universitaria, llha do Fundao 21.910, Rio de Janeiro, RJ, Brasil.

and inhibiting transfer of the phosphoryl group to glucose. Materials and Methods

Yeast hexokinase (ATP:D-hexose 6-phosphotransferase; EC sulfate-free, was purchased as the lyophilized powder from baker's yeast (Sigma Chemicals), which is purified by the method described by Kaji et al. [7]. Hydrolysis of ATP was assayed in a standard medium containing 50 mM Tris-HCl (pH 7.4), 5 mM MgC!2, 1 mM [a2p]ATP, and 0.1 mg/ml yeast hexokinase. The reaction was stopped by addition of trichloroacetic acid to a final concentration of 6%. The rate of hydrolysis was determined by measuring the 32Pi produced from [32p]ATP by cintillation counting [8]. Glucose phosphorylation was assayed in a medium containing 50 mM Tris-HC! (pH 7.4), 1.5 mM MgCl 2, 1.5 mM glucose, 0.5 mM ATP and 0.2/~g/ml hexokinase. The extent of glucose phosphorylation was determined by spectrophotometric measurement of NADH oxidation. To prevent any effect of dimethyl sulfoxide during this assay, the yeast hexokinase reaction was stopped by heating at 100°C for 10 s, and diluted 5-fold in a medium containing 50 mM Tris-HCl (pH 7.0), 5 mM MgC! 2, 50 mM KCI, 3 mM phosphoenolpyruvate, 0.225 mM NADH and 1 unit of a mixture of pyruvate kinase and lactate dehydrogenase.

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Results Yeast hexokinase can catalyze ATP hydrolysis, but at a much slower rate than glucose phosphorylation [9]. Here it is shown (Fig. I) that increasing concentrations of dimethyl sulfoxide activate the rate of hydrolysis of A T P catalyzed by hexokinase. Maximal activation (4.2 fold) occurs at 30% dimethyl sulfoxide. Transfer of the phosphoryl group of A T P to glucose, on the other hand, is inhibited by dimethyl sulfoxide (Fig. 1B). in the presence of 40% dimethyl sulfoxide, the ratio between ATPase activity and glucose phosphorylation increases 10-fold in comparison to that in purely aqueous medium. These results suggest that this organic solvent may change the relative effectiveness with which yeast hexokinase u ~ s water or hexose as phosphoryl acceptor. The sugars, xylose and lyxose, also stimulate the ATPase activity catalyzed by yeast hexokinase, although neither one is phosphorylated by this enzyme [10-13]. Here it is shown that in aqueous medium xylose stimulates the ATPase activity (Fig. 2A) up to 5-fold. Interestingly, A T P hydrolysis is activated further in the presence of 20% dimethyl sulfoxide. On the other hand, inactivation of glucose phosphorylation by xyiosc is only slightly modified by dimethyl sulfoxide (Fig. 2B). It has been shown that preincubation of hexokinase in the presence of x y l o ~ and Mg-ATP, leads to an irreversible phosphorylation of the hexokinase and to Io~ of hexokinase activity [12,13]. In Fig. 3, hexokinase was preincubated in the presence of A T P and MgCl,, and either dimethyl sulfoxide, xylose or both. At different time intervals, phosphorylation of glucose by yeast hexokinase was measured. It is shown (Fig. 3) that preincubation with dimethyl sulfoxidc does not inactivate the enzyme, whereas when xylose is present dur-





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Fig. 2. Effect of xylosc on hexokinase activity. ATPasc activity (A) and glucose phosphorylation (B) were measured as described in Materials and Methods in aqueous solution (o} or in the presence of 2(}~ dimelhyl sulfoxide{o) and increasing concentrations of xylo~. ing preincubation activity falls to 30% of the original value. This control indicates that the effects of dimethyl sulfoxide seen in Figs. 1 and 2 probably do not involve phosphorylation of the enzyme. Discussion A glucose-induced conformational change in hexokinase has been inferred on the basis of crystallographic, spectrometric and kinetic studies [1,4]. It has been proposed that a conformational change is induced upon binding glucose to hexokinase, which excludes contact o f the sugar with water, leaving just the 6-hydroxymethyl group accessible to the solvent. T h e results presented here suggest that changing the hydrophobicity of the medium with dimethyl sulfoxide renders the A T P bound to the hexokinase more available for hydrolysis than in aqueous medium. In the presence of




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OblSO , (~) Fig. I. Effect of dimethyl sulfoxide on the hydrolysisof ATP (A and B) and glucose phosphoqdation (B). In (A) ATP hydrolysis was measured as described in Materials and Methods, in the presence of none (o). 10oA {o). 20% (zx), 30% (A) and 40% (El) dimethyl sulfoxide, in (B) ATP hydrolysis(o) and glucose phosphorylation (o) were measured as described in Materials and Methods. Activitywas normalized to 5 nmol/min mg for ATPase activityand 138 pmol/min mg for glucose phosphotylation.







Time (min) Fig. 3. Effect of hexokinase preincubaUon in the presence of dimethyl sulfoxide, xyloseand ATE Hexokinase was preincubated in a medium containing 50 mM Tris-HCI (pH 7.4), I mM ATP, 5 mM MgCI2, 0.1 mg/ml hexokinase, and either 20% dimethyl sulfoxide (o). 100 mM xylose (,~), or 20% dimethyl sulfoxide and 100 mM xylose (o). At different intervals an aliquot of 20 #I was diluted in l ml of a medium containing 50 mM Tris-HCI (pH 7.4), 0.2 mM ATP, 5 mM MgCI2, 2 mM gluco~, 3 mM phosphoenolpyruvate, 20 mM KCI, 0.240 mM NADH, pyruvale kinase and lactate dchydrogenase, and assayed for glucose phosphorylation.

307 40% dimethyl sulfoxide, the ratio between transfer of the phosphoryi group from ATP to glucose and ATP to water is 10-fold lower than in aqueous medium (Fig. IB). Furthermore, activation of hydrolysis does not lead to inhibition of enzyme activity. It has been proposed that binding of glucose to the enzyme promotes the closing of the two lobes that form the catalytic site [4]. In general, enzymatic activity depends on hydrophobic interactions between the enzyme, its substrates and water. Thus, a change in water structure may alter the enzyme's co~/formation. The change in specificity of the hexokinase in the presence of dimethyl sulfoxide may be due to a change in the flexibility of the enzyme and to a decrease of water activity within the catalytic site which would allow ATP to be hydrolyzed at a faster rate, as has been shown during non-enzymatic hydrolysis of ATP in the presence of organic solvents [14].

Acknowledgements 1 wish to thank Dr. L. de Meis for constant encouragement and support, Dr. A. Gomez-Puyou for his helpful discussions and V.A. Suzano for technical assistance. This work was supported in part by grants from Fundacao Banco do Brasil, Financiadora de Estudos e Projetos, Conselho Nacional de Desenvolvimento Ci-

entifico e Tecnologico (CNPq), and by Funda~ao de Amparo a Pesquisa do Rio de Janeiro (FAPERJ) to Dr. L. de Meis. MML is a recipient of a fellowship from FAPERJ.

References I Anderson, C.M., Zucker, F.H. and Steitz, T.A. (1979) Science 204, 375-380. 2 Bennett, W.S. and Steitz, T.A. (1978) Proc. Natl. Acad. Sci. USA 75, 4848-4852. 3 Mc Donald, R., Steitz, T.A. and Engelman, D.M. (1979) Biochemistry 18. 338-342. 4 Takahashi, K.. Casey, J.L. and Sturtevanl. M. (1981) Biochemistry 20, 4693-4697. 5 The, R. and Hasselbach. W. (1977) Eur. J. Biochem. 74, 611-621. 6 de Meis, L., Martins, O.B. and Aires, E.W. (1980) Biochemistry 19. 4252-4261. 7 KaJi, A., Trayser, K. and Colowick, S. (1961) Ann. N.Y. Acad. Sci. 94, 798-811. 8 de Meis, L. (1088) Methods Enzymol. 157, 190-206. 9 Kaji. A. and Colowick, S.P. (1965) J. Biol. Chem. 24[], 4454-4462. l0 DelaFuente, G. (1970) Eur. J. Biochem. 16, 24[]-243. 1 ! DelaFuente, G., Lagunas, R. and Sols, A. (1970) Eur. J. Biochem. 16. 226-233. 12 Fernandez, R., Herrero, P., Fernandez, M.T. and Moreno. F.J. (1986) Gen. Microbiol. 132, 3467-3472. 13 Menezez, L.C. and Pudles, J. (1976) Eur. J. Biochem. 65.41-47. 14 de Meis, L. and Suzano, V.A. (1988) FEBS Lett. 232, 73-77.

Effect of dimethyl sulfoxide on phosphoryl transfer catalyzed by yeast hexokinase.

Hexokinase is a phosphotransferase that catalyzes phosphoryl transfer from ATP to glucose much more rapidly than the transfer from ATP to water (i.e.,...
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